Urinary proteomics provides a tool to discover new biomarkers for kidney damage. Proteomics is the study of proteomes, which describes the entire protein content of cells and bodily fluids like urine. A major goal is to identify disease biomarkers in urine that can be used for early diagnosis. Urine contains proteins from glomerular filtration and tubular secretion, as well as exosomes containing membrane and intracellular proteins from kidney epithelial cells. Mass spectrometry techniques like CE-MS can analyze the urinary proteome and peptide database to identify biomarkers for conditions like kidney cancer, diabetic nephropathy, and acute transplant rejection.
Therapeutic plasma exchange (TPE) is an extracorporeal blood purification technique used to remove large molecular weight substances from the plasma, such as pathogenic autoantibodies, immune complexes, and myeloma light chains. There are two main methods for TPE - centrifugal plasma separation and membrane plasma separation. TPE aims to remove the target pathogenic substance by exchanging 1-1.5 plasma volumes in each procedure to allow for redistribution between plasma and tissues. Complications are generally minor but can include hypotension, allergic reactions, and in rare cases mortality. TPE has various indications like myasthenia gravis, Guillain-Barré syndrome, and cryoglobulinemia where removal of
This document summarizes guidelines for evaluating kidney function in potential living donors. It recommends estimating glomerular filtration rate (GFR) using serum creatinine and/or cystatin C to assess kidney function. An initial GFR of 90 mL/min/1.73m2 or greater is acceptable for donation, while GFR between 60-89 mL/min/1.73m2 requires individual assessment. Donors with less than 60 mL/min/1.73m2 are not eligible. The guidelines provide criteria for measurement and interpretation of GFR to safely evaluate and select living kidney donors.
This document discusses renal replacement therapy options including dialysis and renal transplantation. It notes that renal transplantation from a live donor has a 94% 5-year survival rate while deceased donor transplantation is 76%. Reasons for using deceased donors include the number of patients awaiting transplant and potential live donors having complications. Deceased donors can be from donation after brain death or donation after cardiac death. The document outlines the process for identifying potential deceased donors, confirming brain death, obtaining consent, notifying the retrieval team, and carefully managing the donor. It describes what constitutes a deceased donor according to medical guidelines. Key steps in the deceased donor organ retrieval process are also summarized.
The patient is a 49-year-old woman with end-stage renal disease and diabetes who presented with altered mental status. She receives hemodialysis three times per week for a few years. Recently, she has been increasingly tired, weak, and unable to perform daily activities with poor appetite and nausea. On examination, she was pale and swollen with low hemoglobin. Tests found elevated creatinine, BUN, and electrolyte abnormalities. The most probable diagnosis is inadequate hemodialysis, as her symptoms and labs are consistent with worsening uremia due to insufficient solute clearance from her dialysis sessions. Kt/V is a measure of dialysis adequacy that accounts for urea clearance and patient
The document discusses Patient Blood Management (PBM), which is a multidisciplinary approach to optimize care for patients who may need blood transfusions. It involves strategies before, during, and after surgery/procedures to minimize blood loss and transfusions. Key preoperative strategies include identifying and treating anemia, assessing bleeding risk, and considering preoperative autologous blood donation. Intraoperative strategies focus on techniques to reduce blood loss like cell salvage and tranexamic acid. Postoperative care emphasizes continued efforts to minimize blood loss and optimize physiology. The overall goals are to improve patient outcomes, reduce costs, and ensure an adequate blood supply.
The CDC crossmatch is the traditional method for detecting donor-specific HLA antibodies before transplantation. It detects complement-fixing antibodies but has low sensitivity and specificity. Modifications like adding anti-human globulin increase its sensitivity but it still has limitations. More sensitive techniques like flow cytometry crossmatch, solid phase immunoassays like Luminex single antigen beads, and virtual crossmatches based on donor HLA profiles are now commonly used before transplantation to better assess antibody risk. Performing multiple complementary tests provides the most accurate assessment of donor-specific antibody levels and risk.
Therapeutic plasma exchange (TPE) is an extracorporeal blood purification technique used to remove large molecular weight substances from the plasma, such as pathogenic autoantibodies, immune complexes, and myeloma light chains. There are two main methods for TPE - centrifugal plasma separation and membrane plasma separation. TPE aims to remove the target pathogenic substance by exchanging 1-1.5 plasma volumes in each procedure to allow for redistribution between plasma and tissues. Complications are generally minor but can include hypotension, allergic reactions, and in rare cases mortality. TPE has various indications like myasthenia gravis, Guillain-Barré syndrome, and cryoglobulinemia where removal of
This document summarizes guidelines for evaluating kidney function in potential living donors. It recommends estimating glomerular filtration rate (GFR) using serum creatinine and/or cystatin C to assess kidney function. An initial GFR of 90 mL/min/1.73m2 or greater is acceptable for donation, while GFR between 60-89 mL/min/1.73m2 requires individual assessment. Donors with less than 60 mL/min/1.73m2 are not eligible. The guidelines provide criteria for measurement and interpretation of GFR to safely evaluate and select living kidney donors.
This document discusses renal replacement therapy options including dialysis and renal transplantation. It notes that renal transplantation from a live donor has a 94% 5-year survival rate while deceased donor transplantation is 76%. Reasons for using deceased donors include the number of patients awaiting transplant and potential live donors having complications. Deceased donors can be from donation after brain death or donation after cardiac death. The document outlines the process for identifying potential deceased donors, confirming brain death, obtaining consent, notifying the retrieval team, and carefully managing the donor. It describes what constitutes a deceased donor according to medical guidelines. Key steps in the deceased donor organ retrieval process are also summarized.
The patient is a 49-year-old woman with end-stage renal disease and diabetes who presented with altered mental status. She receives hemodialysis three times per week for a few years. Recently, she has been increasingly tired, weak, and unable to perform daily activities with poor appetite and nausea. On examination, she was pale and swollen with low hemoglobin. Tests found elevated creatinine, BUN, and electrolyte abnormalities. The most probable diagnosis is inadequate hemodialysis, as her symptoms and labs are consistent with worsening uremia due to insufficient solute clearance from her dialysis sessions. Kt/V is a measure of dialysis adequacy that accounts for urea clearance and patient
The document discusses Patient Blood Management (PBM), which is a multidisciplinary approach to optimize care for patients who may need blood transfusions. It involves strategies before, during, and after surgery/procedures to minimize blood loss and transfusions. Key preoperative strategies include identifying and treating anemia, assessing bleeding risk, and considering preoperative autologous blood donation. Intraoperative strategies focus on techniques to reduce blood loss like cell salvage and tranexamic acid. Postoperative care emphasizes continued efforts to minimize blood loss and optimize physiology. The overall goals are to improve patient outcomes, reduce costs, and ensure an adequate blood supply.
The CDC crossmatch is the traditional method for detecting donor-specific HLA antibodies before transplantation. It detects complement-fixing antibodies but has low sensitivity and specificity. Modifications like adding anti-human globulin increase its sensitivity but it still has limitations. More sensitive techniques like flow cytometry crossmatch, solid phase immunoassays like Luminex single antigen beads, and virtual crossmatches based on donor HLA profiles are now commonly used before transplantation to better assess antibody risk. Performing multiple complementary tests provides the most accurate assessment of donor-specific antibody levels and risk.
This document discusses prescribing acute and chronic peritoneal dialysis. For acute PD, it recommends using a Tenckhoff catheter and automated cyclers. Exchanges should be hourly with 2L volumes. Clearance is monitored using BUN levels and D:P ratios. Complications include abdominal distention and peritonitis. For chronic PD, clearance targets are a Kt/V of 1.7 per week. Prescriptions are based on residual renal function, transporter status, and body size. CAPD and APD are both options depending on lifestyle. Clearance can be increased by optimizing exchange volumes, frequency, and solution tonicity.
This document outlines the plasmapheresis protocol at the New Mansoura General Hospital Nephrology Department in Egypt. It defines plasmapheresis as removing, treating, and returning blood plasma from circulation. Examples of diseases treated with plasmapheresis include idiopathic pulmonary fibrosis, Guillain-Barre syndrome, and thrombotic thrombocytopenic purpura. The procedure, complications, replacement solutions, and post-procedure care are described.
Therapeutic plasma exchange (TPE) is an extracorporeal treatment that removes plasma and pathogenic substances like antibodies, immune complexes, or large molecules from the plasma. During TPE, whole blood is separated into components by centrifugation and the plasma is removed and discarded while cellular elements are returned to the patient mixed with a replacement fluid. Early studies found that TPE plus standard therapy for multiple myeloma patients with acute kidney injury improved renal recovery rates compared to standard therapy alone, though larger subsequent studies found no difference in outcomes. TPE is effective at removing various pathogenic factors from circulation that cause diseases like Guillain-Barré syndrome, antibody-mediated transplant rejection, systemic lupus erythematosus, and
This document discusses anticoagulation during hemodialysis. It begins by explaining coagulation and anticoagulants. It then discusses how hemodialysis can activate coagulation pathways due to interactions with artificial surfaces. Unfractionated heparin is most commonly used for anticoagulation during dialysis due to its low cost and short half-life, though it carries bleeding risks. Low molecular weight heparins are also used and provide benefits like less bleeding risk. Anticoagulation is monitored during dialysis through tests like aPTT and ACT to prevent clotting while limiting bleeding risks.
The document summarizes the evaluation of an adult kidney transplant recipient. It discusses timing transplantation based on GFR levels, screening for contraindications like infections and cardiovascular disease, evaluating immunological factors like PRA and HLA typing, and special considerations for populations like diabetics, children, and those on dialysis. The goal of the evaluation is to minimize risks and maximize outcomes for the recipient and longevity of the transplanted kidney.
metabolic acidosis develops because of defects in the ability of the renal tubules to perform the normal functions required to maintain acid-base balance.
hepatorenal syndrome is a one of the complication of cirrhosis of liver. It causes hepatic decompensation of liver. It has high risk of mortality. HRS has two types and type 1 usually present as a acute kidney injury. so, at first HRS should exclude from AKI. HRS type 2 present as a refractory ascites. As this has worst prognosis, only valuable management is liver transplantation.
The document discusses renal failure and its relationship to cardiovascular disease. It provides statistics on the prevalence, incidence, and mortality rates of renal failure in the US and Australia. It then covers topics such as the structure and function of the kidneys, classification of renal failure, its effects on the cardiovascular system, and approaches to treatment including dialysis, transplantation, and their risks.
leucodepletion is the removal of 99% leucocytes from the whole blood, pcv or platelets before transfusing into the donor.
this process many infections, transfusion reactions..
This document discusses concepts related to organ transplantation and immunology. It covers recognition of danger signals by the immune system through pattern recognition receptors on antigen presenting cells. It also describes major histocompatibility complexes and their role in antigen presentation to T cells. Finally, it mentions early inflammatory responses, ischemia-reperfusion injury, and alloimmune responses involved in organ transplantation and rejection.
This document discusses the management of anemia in chronic kidney disease (CKD). It begins by defining anemia and its causes in CKD, which include reduced erythropoietin production and decreased red blood cell survival due to kidney failure. Left untreated, anemia in CKD can lead to deterioration in cardiac function, impaired cognition, and increased fatigue and mortality risk. The main therapeutic options for treating anemia in CKD are red blood cell transfusions, androgens, and erythropoiesis-stimulating agents (ESAs). ESAs such as epoetin alfa and darbepoetin alfa are now the standard treatment as they reduce transfusion needs and risks while helping to mobilize
This document summarizes a presentation on therapeutic plasma exchange (PEX) given by Kamal Mohamed Okasha. It provides an overview of the PEX procedure and potential indications for PEX, including Goodpasture's Syndrome, thrombotic thrombocytopenic purpura, cryoglobulinemia, multiple myeloma, and ANCA disease. It discusses complications of PEX and guidelines for efficacy based on recent studies. In particular, it examines the use of PEX for Goodpasture's Syndrome, noting that PEX aims to remove circulating anti-GBM antibodies and that studies have found improved outcomes, including renal function and survival, for patients receiving PEX treatment.
Anemia of renal disease is common and is associated with significant morbidity and death. It is mainly caused by a decrease in erythropoietin production in the kidneys and can be partially corrected with erythropoiesis-stimulating agents (ESAs). However, randomized controlled trials have shown that using ESAs to target normal hemoglobin levels can be harmful, and have called into question any benefits of ESA treatment other than avoidance of transfusions.
Basic plasmapheresis prof. dr. montasser zeidFarragBahbah
1. Plasmapheresis involves removing plasma from a patient and replacing it with either fresh frozen or stored plasma. It can remove pathogenic factors like antibodies, immune complexes, and proteins.
2. There are two main techniques for plasmapheresis - centrifugal separation and membrane plasmafiltration. Complications can include hypotension, bleeding, and allergic reactions.
3. Plasmapheresis is used to treat various conditions and is categorized based on evidence. It may be used as an adjunctive therapy for sepsis to remove harmful molecules.
This document discusses different techniques for autologous blood transfusion including pre-operative blood donation, acute normovolaemic haemodilution, and intra-operative and post-operative blood salvage. Pre-operative blood donation allows collection of blood 4-5 weeks before surgery while acute normovolaemic haemodilution involves removing blood just before or after anesthesia and replacing it with fluids. Intra-operative and post-operative blood salvage uses equipment to collect and filter blood from the surgical site for reinfusion. Each technique has advantages and appropriate applications depending on the surgical situation and patient factors. Together, autologous transfusion techniques can significantly reduce the need for allogenic blood transfusions
The document discusses complications of peritoneal dialysis, specifically peritonitis. It describes the typical presentation of peritonitis as abdominal pain and cloudy dialysate fluid. Causes include breaks in sterile technique or recent infections. Diagnosis requires abdominal pain and cloudy fluid with leukocytosis. Treatment involves empiric antibiotics targeting gram positive and negative organisms. Outcomes depend on causative organisms and whether the peritoneal catheter is infected.
Hepatorenal syndrome is a condition characterized by impaired renal function in patients with advanced liver disease and portal hypertension. There are two types - type 1 is rapid and progressive, leading to death within a month without treatment, while type 2 is less severe but still associated with worse prognosis. The pathogenesis involves splanchnic vasodilation triggering renal vasoconstriction. Treatment involves vasoconstrictors like terlipressin combined with albumin to increase mean arterial pressure and improve renal function. Achieving at least a 10 mmHg increase in MAP with vasoconstrictor therapy correlates with better renal outcomes in hepatorenal syndrome patients.
Dr. Kumar presented on renal replacement therapy. The key points are:
1. Approximately 5% of critically ill patients with AKI will require RRT, with a mortality rate as high as 60%.
2. RRT options include intermittent HD, continuous therapies like CVVH/CVVHD/CVVHDF, and SLED.
3. The choice of RRT depends on the patient's cardiovascular status, resources available, and whether fluid removal or solute clearance is required. CRRT is preferred for hemodynamically unstable patients.
Urinary proteomics has potential to discover new biomarkers for kidney damage. Proteomics analyzes entire proteomes and can identify dynamic changes in protein profiles from stimuli. Biomarker discovery in urine is promising due to non-invasive large sample collection. Urinary exosomes contain proteins that can mark renal dysfunction and injury from all nephron segments. Mass spectrometry aids characterization of urinary peptides and proteins, helping diagnose conditions like cancer, glomerular diseases, transplant rejection, and predict kidney operation need. Multiple biomarkers may better diagnose complex diseases than single markers.
Safety pharmacology aims to identify potential adverse effects of new drugs prior to clinical trials. It involves evaluating a drug's effects on major organ systems like the cardiovascular, central nervous, and respiratory systems. Biomarkers and newer approaches can provide mechanistic insight. Renal safety assessment is important and may involve in vivo, in vitro, and in silico models. Biomarkers of kidney injury like KIM-1 and clusterin are being used. Safety pharmacology helps predict hazards, identify risks, and facilitate risk management of new drugs.
This document discusses prescribing acute and chronic peritoneal dialysis. For acute PD, it recommends using a Tenckhoff catheter and automated cyclers. Exchanges should be hourly with 2L volumes. Clearance is monitored using BUN levels and D:P ratios. Complications include abdominal distention and peritonitis. For chronic PD, clearance targets are a Kt/V of 1.7 per week. Prescriptions are based on residual renal function, transporter status, and body size. CAPD and APD are both options depending on lifestyle. Clearance can be increased by optimizing exchange volumes, frequency, and solution tonicity.
This document outlines the plasmapheresis protocol at the New Mansoura General Hospital Nephrology Department in Egypt. It defines plasmapheresis as removing, treating, and returning blood plasma from circulation. Examples of diseases treated with plasmapheresis include idiopathic pulmonary fibrosis, Guillain-Barre syndrome, and thrombotic thrombocytopenic purpura. The procedure, complications, replacement solutions, and post-procedure care are described.
Therapeutic plasma exchange (TPE) is an extracorporeal treatment that removes plasma and pathogenic substances like antibodies, immune complexes, or large molecules from the plasma. During TPE, whole blood is separated into components by centrifugation and the plasma is removed and discarded while cellular elements are returned to the patient mixed with a replacement fluid. Early studies found that TPE plus standard therapy for multiple myeloma patients with acute kidney injury improved renal recovery rates compared to standard therapy alone, though larger subsequent studies found no difference in outcomes. TPE is effective at removing various pathogenic factors from circulation that cause diseases like Guillain-Barré syndrome, antibody-mediated transplant rejection, systemic lupus erythematosus, and
This document discusses anticoagulation during hemodialysis. It begins by explaining coagulation and anticoagulants. It then discusses how hemodialysis can activate coagulation pathways due to interactions with artificial surfaces. Unfractionated heparin is most commonly used for anticoagulation during dialysis due to its low cost and short half-life, though it carries bleeding risks. Low molecular weight heparins are also used and provide benefits like less bleeding risk. Anticoagulation is monitored during dialysis through tests like aPTT and ACT to prevent clotting while limiting bleeding risks.
The document summarizes the evaluation of an adult kidney transplant recipient. It discusses timing transplantation based on GFR levels, screening for contraindications like infections and cardiovascular disease, evaluating immunological factors like PRA and HLA typing, and special considerations for populations like diabetics, children, and those on dialysis. The goal of the evaluation is to minimize risks and maximize outcomes for the recipient and longevity of the transplanted kidney.
metabolic acidosis develops because of defects in the ability of the renal tubules to perform the normal functions required to maintain acid-base balance.
hepatorenal syndrome is a one of the complication of cirrhosis of liver. It causes hepatic decompensation of liver. It has high risk of mortality. HRS has two types and type 1 usually present as a acute kidney injury. so, at first HRS should exclude from AKI. HRS type 2 present as a refractory ascites. As this has worst prognosis, only valuable management is liver transplantation.
The document discusses renal failure and its relationship to cardiovascular disease. It provides statistics on the prevalence, incidence, and mortality rates of renal failure in the US and Australia. It then covers topics such as the structure and function of the kidneys, classification of renal failure, its effects on the cardiovascular system, and approaches to treatment including dialysis, transplantation, and their risks.
leucodepletion is the removal of 99% leucocytes from the whole blood, pcv or platelets before transfusing into the donor.
this process many infections, transfusion reactions..
This document discusses concepts related to organ transplantation and immunology. It covers recognition of danger signals by the immune system through pattern recognition receptors on antigen presenting cells. It also describes major histocompatibility complexes and their role in antigen presentation to T cells. Finally, it mentions early inflammatory responses, ischemia-reperfusion injury, and alloimmune responses involved in organ transplantation and rejection.
This document discusses the management of anemia in chronic kidney disease (CKD). It begins by defining anemia and its causes in CKD, which include reduced erythropoietin production and decreased red blood cell survival due to kidney failure. Left untreated, anemia in CKD can lead to deterioration in cardiac function, impaired cognition, and increased fatigue and mortality risk. The main therapeutic options for treating anemia in CKD are red blood cell transfusions, androgens, and erythropoiesis-stimulating agents (ESAs). ESAs such as epoetin alfa and darbepoetin alfa are now the standard treatment as they reduce transfusion needs and risks while helping to mobilize
This document summarizes a presentation on therapeutic plasma exchange (PEX) given by Kamal Mohamed Okasha. It provides an overview of the PEX procedure and potential indications for PEX, including Goodpasture's Syndrome, thrombotic thrombocytopenic purpura, cryoglobulinemia, multiple myeloma, and ANCA disease. It discusses complications of PEX and guidelines for efficacy based on recent studies. In particular, it examines the use of PEX for Goodpasture's Syndrome, noting that PEX aims to remove circulating anti-GBM antibodies and that studies have found improved outcomes, including renal function and survival, for patients receiving PEX treatment.
Anemia of renal disease is common and is associated with significant morbidity and death. It is mainly caused by a decrease in erythropoietin production in the kidneys and can be partially corrected with erythropoiesis-stimulating agents (ESAs). However, randomized controlled trials have shown that using ESAs to target normal hemoglobin levels can be harmful, and have called into question any benefits of ESA treatment other than avoidance of transfusions.
Basic plasmapheresis prof. dr. montasser zeidFarragBahbah
1. Plasmapheresis involves removing plasma from a patient and replacing it with either fresh frozen or stored plasma. It can remove pathogenic factors like antibodies, immune complexes, and proteins.
2. There are two main techniques for plasmapheresis - centrifugal separation and membrane plasmafiltration. Complications can include hypotension, bleeding, and allergic reactions.
3. Plasmapheresis is used to treat various conditions and is categorized based on evidence. It may be used as an adjunctive therapy for sepsis to remove harmful molecules.
This document discusses different techniques for autologous blood transfusion including pre-operative blood donation, acute normovolaemic haemodilution, and intra-operative and post-operative blood salvage. Pre-operative blood donation allows collection of blood 4-5 weeks before surgery while acute normovolaemic haemodilution involves removing blood just before or after anesthesia and replacing it with fluids. Intra-operative and post-operative blood salvage uses equipment to collect and filter blood from the surgical site for reinfusion. Each technique has advantages and appropriate applications depending on the surgical situation and patient factors. Together, autologous transfusion techniques can significantly reduce the need for allogenic blood transfusions
The document discusses complications of peritoneal dialysis, specifically peritonitis. It describes the typical presentation of peritonitis as abdominal pain and cloudy dialysate fluid. Causes include breaks in sterile technique or recent infections. Diagnosis requires abdominal pain and cloudy fluid with leukocytosis. Treatment involves empiric antibiotics targeting gram positive and negative organisms. Outcomes depend on causative organisms and whether the peritoneal catheter is infected.
Hepatorenal syndrome is a condition characterized by impaired renal function in patients with advanced liver disease and portal hypertension. There are two types - type 1 is rapid and progressive, leading to death within a month without treatment, while type 2 is less severe but still associated with worse prognosis. The pathogenesis involves splanchnic vasodilation triggering renal vasoconstriction. Treatment involves vasoconstrictors like terlipressin combined with albumin to increase mean arterial pressure and improve renal function. Achieving at least a 10 mmHg increase in MAP with vasoconstrictor therapy correlates with better renal outcomes in hepatorenal syndrome patients.
Dr. Kumar presented on renal replacement therapy. The key points are:
1. Approximately 5% of critically ill patients with AKI will require RRT, with a mortality rate as high as 60%.
2. RRT options include intermittent HD, continuous therapies like CVVH/CVVHD/CVVHDF, and SLED.
3. The choice of RRT depends on the patient's cardiovascular status, resources available, and whether fluid removal or solute clearance is required. CRRT is preferred for hemodynamically unstable patients.
Urinary proteomics has potential to discover new biomarkers for kidney damage. Proteomics analyzes entire proteomes and can identify dynamic changes in protein profiles from stimuli. Biomarker discovery in urine is promising due to non-invasive large sample collection. Urinary exosomes contain proteins that can mark renal dysfunction and injury from all nephron segments. Mass spectrometry aids characterization of urinary peptides and proteins, helping diagnose conditions like cancer, glomerular diseases, transplant rejection, and predict kidney operation need. Multiple biomarkers may better diagnose complex diseases than single markers.
Safety pharmacology aims to identify potential adverse effects of new drugs prior to clinical trials. It involves evaluating a drug's effects on major organ systems like the cardiovascular, central nervous, and respiratory systems. Biomarkers and newer approaches can provide mechanistic insight. Renal safety assessment is important and may involve in vivo, in vitro, and in silico models. Biomarkers of kidney injury like KIM-1 and clusterin are being used. Safety pharmacology helps predict hazards, identify risks, and facilitate risk management of new drugs.
Generation of Induced Pluripotent Stem Cells from Urine Cell Derived from Pat...Charles Malcolm Roberson
This study aimed to generate induced pluripotent stem cells (iPSCs) from urine cells of patients with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder. Urine samples were collected from three patients, cultured, and characterized. The cells were then to be reprogrammed into iPSCs via electroporation of plasmids containing reprogramming factors. While initial culturing of control urine cells was unsuccessful, culturing of cells from a second FOP patient showed promise. Future work includes characterizing the urine cells and reprogramming them to produce patient-specific iPSCs for studying FOP.
This document discusses hematological investigations that are useful in clinical dentistry. It begins by explaining that laboratory studies provide information to identify the nature of diseases by examining tissues, blood, and other specimens. Some key points covered include:
- Laboratory tests can confirm or reject clinical diagnoses and provide guidance for patient management.
- Tests are classified as screening or diagnostic based on their sensitivity and specificity.
- Common hematological investigations performed include complete blood count, hemoglobin analysis, and coagulation tests.
- Proper collection and preservation of blood samples is important for accurate test results.
So in summary, the document outlines the role of laboratory hematological tests in clinical dentistry for diagnosing systemic conditions and
Nephrotic syndrome in Sickle Cell Disease of Western Odisha, India: A case re...inventionjournals
Sickle cell disease causes a distinct pattern of glomerular dysfunction. Subjects with sickle cell disease (SCD) are known to develop many potential functional and structural renal abnormalities. Glomerular hypertension and hyper filtration are thought to play a major role in the development of glomerular disease in subjects with SCD. We reported 5 unusual cases of sickle cell disease presenting as nephrotic syndrome. KEYWORDS- Nephrotic syndrome, sickle cell disease
Nephrotic syndrome in Sickle Cell Disease of Western Odisha, India: A case re...inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
The document provides an overview of renal anatomy and physiology. It describes the microscopic structure of the nephron, including the glomerulus and its role in filtering blood. The major functions of the kidneys are also summarized, such as regulating fluid balance and electrolytes, producing hormones, and eliminating waste. Key anatomical structures involved in these functions are the glomerulus, proximal and distal tubules, as well as the surrounding blood vessel network that allows for reabsorption and secretion.
The document discusses the process of erythropoiesis, or red blood cell formation, including the regulation of erythropoiesis through erythropoietin (EPO) production and feedback loops to control hemoglobin levels. It describes the multi-step differentiation process from hematopoietic stem cells in bone marrow to mature red blood cells, and the roles of the liver and kidney in producing EPO at different developmental stages. The document also outlines the metabolic pathways in red blood cells, their lifespan of around 120 days before removal by the spleen, and the breakdown of hemoglobin into bilirubin to be processed and excreted by the liver and intest
This document provides an overview of urological laboratory investigations, focusing on urine analysis. It discusses the various types of urine samples and how to collect and transport specimens. The document describes microscopic and biochemical examinations of urine to detect abnormalities. Specific tests are covered, including urine cytology to detect urothelial cancer and markers like bladder tumor antigen. Interpretation of urine analysis results and limitations of certain tests are also summarized.
A basic and worth information for diagnostic is urine microscopy. ideally it should be by the physician at his clinic to add and correlate diagnosis promptly. this will make physician confident in dealing with patients. it also help in follow up the consequences in some important glomerulopathies.
Acute kidney injury (AKI) is a serious condition with high mortality and costs that has seen increasing incidence but little improvement in outcomes. Proteomic analysis of urine and blood can help identify biomarker patterns that enable early AKI diagnosis, distinguish AKI subtypes and etiologies, and provide insights into disease mechanisms. Studies have found biomarkers such as NGAL, annexin A5, 6-PGLS, protein S100-P, CALCA, CALR, CA12, CLEC1A, PTK7, KIM-1, NPPC, NUCB2 and PGF that are elevated early in preterm infants and sepsis patients who develop AKI. Proteomic profiling of
Components of blood and blood clotting mechanismPritamCahtterjee
This document provides an overview of the components of blood and the blood clotting mechanism. It discusses the properties and composition of blood, the constituents of plasma including plasma proteins, red blood cells, white blood cells, and platelets. It then describes the process of hemostasis and blood coagulation, how a blood clot is formed, and various tests for blood clotting. It also reviews different anticoagulant drugs and considerations for managing patients on anticoagulant therapy in dental practice.
This document discusses uromodulin (UMOD), a glycoprotein expressed in the kidney that is the most abundant protein in urine. It summarizes the discovery of UMOD, its normal biosynthesis and function anchoring to the kidney epithelial cell membrane and release into urine. Mutations in UMOD can cause storage diseases with progressive kidney failure due to misfolded protein accumulation. High urine or serum UMOD levels are associated with chronic kidney disease and may stimulate inflammation through binding immune cells. Downregulating UMOD synthesis may help slow chronic kidney disease progression. Biochemical analysis methods for detecting and quantifying UMOD in various samples are also summarized.
This case presentation describes a 52-year-old male patient who presented with vomiting, hiccups, and low back pain. Laboratory tests revealed anemia, renal failure, and abnormal calcium, phosphate, and albumin levels. Imaging showed lytic bone lesions and cysts on the kidneys. A kidney biopsy showed acute tubular injury. Tests confirmed a diagnosis of multiple myeloma. Multiple myeloma is a blood cancer developing in the bone marrow, with risk factors including genetic predisposition and occupational exposures. The patient is being treated with chemotherapy, steroids, and fluid management for his condition.
This document provides information about analyzing cerebrospinal fluid (CSF). It discusses the formation and function of CSF and describes routine laboratory assays performed on CSF samples, including examinations of appearance, cell counts, chemical analysis, and microbiology. Procedures for collecting and handling CSF samples are outlined. Normal ranges and pathological conditions associated with abnormal CSF results are also reviewed.
This document provides an overview of leucopoiesis, the process by which white blood cells are produced. It discusses the classification, morphology, properties, functions, and applied physiology of the main white blood cell types - granulocytes like neutrophils, eosinophils and basophils, and agranulocytes like lymphocytes and monocytes. It also covers pathological variations in white blood cell counts, disorders like cyclic neutropenia and hypereosinophilic syndrome, and recent advances in areas like bone marrow transplantation and flow cytometry.
This document provides an overview of leucopoiesis, the process by which white blood cells are produced. It discusses the classification, morphology, properties, functions, and applied physiology of the main white blood cell types - granulocytes like neutrophils, eosinophils and basophils, and agranulocytes like lymphocytes and monocytes. It also covers pathological variations in white blood cell counts, disorders like cyclic neutropenia and hypereosinophilic syndrome, and recent advances in areas like bone marrow transplantation and flow cytometry.
Review (ca 2007) of Uremic Toxins Accumulating in Patients with Chronic and End Stage Renal Disease modified from a presentation I gave in Fellow's Grand rounds.
Relied heavily on publications from the EU Toxin Work Group Work, which provides more up to date information:
http://www.uremic-toxins.org/
The document discusses overactive bladder (OAB) and its treatment. It defines OAB as a condition caused by involuntary bladder contractions resulting in urgency and frequent urination. Common causes include neurological issues, bladder problems, medications, and idiopathic factors. Treatment involves pharmacotherapy using anticholinergic drugs to suppress contractions, bladder retraining, or surgery in severe cases. Lifestyle changes like limiting caffeine and bladder retraining exercises can also help manage symptoms.
Similar to THE URINARY PROTEOMICS:A TOOL TODISCOVER NEW AND POTENT BIOMARKERS FORKIDNEY DAMAGE (20)
stroke biomarkers a new era with diagnostic promise.pptxMoustafa Rezk
1. Several biomarkers have potential for diagnosing and predicting outcomes in stroke patients. Biomarkers of brain injury like S100B, MMP-9, NSE, and GFAP are elevated after stroke and can differentiate between ischemic and hemorrhagic stroke. Higher levels generally indicate larger infarct size and worse prognosis.
2. MicroRNAs in plasma also show promise as stroke biomarkers. Certain microRNAs are differentially regulated after ischemic stroke and correlate with conditions like hemorrhage.
3. Biomarkers of inflammation like IL-6, procalcitonin, and TNF-a levels increase after stroke and may provide some diagnostic or prognostic information, though more research is still needed.
Adipokines in Insulin Resistance Current Updates.pptxMoustafa Rezk
This document summarizes the current understanding of adipokines and their role in insulin resistance. It begins by describing adipose tissue as an endocrine organ that secretes adipokines, which can regulate fat mass and insulin resistance. It then discusses several specific adipokines (adiponectin, omentin, visfatin, leptin, resistin, lipocalin-2, retinol binding protein-4, chemerin, TNF-α, IL-6) and how each contributes to insulin resistance through different mechanisms like decreasing insulin receptor activity or modulating glucose transport. Overall, dysfunctional adipose tissue secretion of altered adipokine levels is associated with insulin resistance and related chronic diseases.
Inappropriate use of laboratory resources.pptxMoustafa Rezk
This document discusses inappropriate use of laboratory resources and provides strategies to overcome it. It finds that 60-70% of high-throughput laboratory tests may be inappropriate or of minor clinical relevance. Reasons for inappropriate testing include easy online ordering, defensive medicine, and lack of education on test value. Consequences include incorrect diagnoses, unnecessary follow-up tests, and increased costs. Strategies to address this include educational interventions, limiting tests through algorithms, and demand management like enforcing re-testing intervals and streamlining test panels. When correctly implemented with clinician approval, demand management can optimize testing to benefit patients, clinicians and hospitals.
Adipokines as a potential biomarkers for vascular complications in type 2 dia...Moustafa Rezk
Adipose tissue has come into focus as an endocrine organAdipose tissue secretes a variety of bioactive peptides (adipokines).Adipokines may locally regulate fat mass by modulating adipocyte size/number or angiogenesis and inversely increased fat mass leads to dysregulation of adipocyte functions.
Impact of Laboratory Automation on quality and TRT. Evaluating and Selecting...Moustafa Rezk
The document discusses laboratory automation and provides recommendations for clinical labs evaluating their need for automation. It begins with defining automation and providing examples of modular pre-analytic and task-targeted automation systems that do not involve tracks and robotics. The document then recommends labs map their workflow and identify bottlenecks before considering automation solutions. It emphasizes optimizing current processes and provides examples of total lab automation systems from various vendors.
stroke biomarkers a new era with diagnostic promise.pptxMoustafa Rezk
1. The document discusses various potential biomarkers for diagnosing stroke.
2. Biomarkers discussed include proteins indicating brain injury like S100β, MMP-9, and NSE; inflammation markers like interleukin-6, procalcitonin, and TNF-α; and oxidative stress biomarkers like thioredoxin and F2-isoprostanes.
3. Additional biomarkers discussed are lipids such as apolipoprotein A-1 and fatty acid binding protein 4, which have shown potential for distinguishing between ischemic and hemorrhagic stroke.
Maldi tof-ms analysis in identification of prostate cancerMoustafa Rezk
MALDI-TOF-MS analysis was used to generate proteomic profiles from plasma samples to identify biomarkers for prostate cancer. Samples were prepared using magnetic beads to separate proteins, then analyzed using MALDI-TOF-MS. Bioinformatics tools were used to generate classification models to distinguish prostate cancer patients from healthy controls based on differences in peak intensities. A 5-peak model achieved 87.5% sensitivity and 92.9% specificity. The study demonstrated the potential of MALDI-TOF proteomic profiling for early prostate cancer screening and diagnosis in Egypt. Proteomic biomarkers may help reduce unnecessary biopsies and stratify patients in the future.
The document discusses acute kidney injury (AKI) in patients with COVID-19. It finds that AKI occurs in 2.9-5.1% of COVID-19 patients and up to 27.8% in severe cases. AKI risk is higher in those with elevated baseline creatinine or comorbidities. SARS-CoV-2 can directly infect kidney cells, causing tubular necrosis and inflammation. Early AKI detection using biomarkers can help allocate resources and serial measurements may predict AKI during ICU stays. Protecting kidney function in COVID-19 patients, especially with AKI, through treatments like CRRT may help prevent fatalities.
Biomarkers for Depression: Recent InsightsMoustafa Rezk
This document summarizes recent research on biomarkers for depression. It discusses how the COVID-19 pandemic has increased rates of depression and how the virus can damage the brain. Potential biological markers for depression include changes in gray matter volume, the circadian cycle, and levels of hormones, neurotransmitters, cytokines and other biomarkers. The document then presents a novel potential peripheral biomarker involving the G protein Gsα, which is involved in cAMP signaling. Studies found Gsα was more localized to lipid rafts in the blood cells of depressed individuals, correlated with lower cAMP production. Treatment responded showed a shift of Gsα out of lipid rafts and increased cAMP signaling, suggesting this biomarker could help predict antidepressant response.
Recent adipokines as a markers of diabetic complicationsMoustafa Rezk
1) The document discusses recent adipokines such as apelin, visfatin, and omentin-1 as markers of diabetic complications. It provides information on their roles and levels in diabetes and related conditions.
2) Apelin levels are reduced in newly diagnosed diabetes but elevated in diabetes with complications like nephropathy. Visfatin levels correlate with insulin resistance and inflammation.
3) Omentin-1 appears protective against cardiovascular disease as it induces vasodilation and lowers inflammation. Studies found lower levels in diabetes patients with macrovascular complications.
This document discusses novel biomarkers for the diagnosis of ovarian carcinoma. It covers gene-based biomarkers like inherited mutations in BRCA1, BRCA2, and DNA mismatch repair genes. Epigenetic changes like DNA methylation and histone modifications in tumor suppressor genes are also discussed. For protein-based biomarkers, the document outlines proteomic pattern diagnostics using mass spectrometry techniques, as well as single biomarker panels like OVA1 that include CA-125, beta-2 microglobulin, transferrin, apolipoprotein A1, and transthyretin. While no single biomarker currently exists for early detection of ovarian cancer, these emerging approaches show promise as non-invasive diagnostic tools.
Ngal ,cystatin c versus creatinine clearence asMoustafa Rezk
This document discusses biomarkers for acute kidney injury (AKI), specifically NGAL and cystatin C. It provides background on the definition and classification of AKI, limitations of creatinine as a biomarker, and the need for earlier detection. It summarizes studies showing NGAL and cystatin C can increase within hours after cardiac surgery or injury, earlier than rises in creatinine. These biomarkers may allow for earlier diagnosis and intervention to prevent further kidney damage.
1. The document discusses recent biomarkers for stroke, including biomarkers of oxidative damage, inflammation, brain injury, lipids, and thrombosis. Biomarkers like F2-isoprostanes, IL-6, miRNAs, S100β, NSE, apo A-1, and FABP4 show potential for diagnosing stroke, determining stroke severity, or distinguishing between stroke types.
2. A panel of 5 biomarkers (apo A-1, PON1, IL-6, TNF-α, and IP-10) showed improved accuracy over single biomarkers for detecting acute stroke versus stroke mimics.
3. Biomarkers may provide objective measures for clinical trials, drug development, and
Laboratory Assessment Of Metabolic DisordersMoustafa Rezk
This document discusses laboratory assessment of metabolic disorders, including disorders of carbohydrate, lipid, protein, mineral, and bone metabolism. It provides definitions of diabetes mellitus and criteria for diagnosis. It also discusses the role of the laboratory in diagnosing and managing diabetes, including markers of acute and chronic control. Additionally, it covers laboratory assessment of disorders of lipid, protein, mineral, and bone metabolism.
This document discusses electrolyte disorders and fluid compartments in the body. It describes that total body water is divided between intracellular and extracellular fluid, separated by semipermeable membranes. Electrolyte levels are tightly regulated between fluid compartments. Common electrolyte disorders like hyponatremia, hypernatremia, hypokalemia, and hyperkalemia are outlined along with their causes, clinical presentations, and treatment approaches.
DIFFICULTIES IN LAB. DIAGNOSIS OF THYROID DISEASEMoustafa Rezk
The document discusses common thyroid diseases and laboratory tests used in their diagnosis. It describes the main thyroid diseases as hypothyroidism, hyperthyroidism, goiters, thyroiditis, solitary thyroid nodules, and cancer. For each disease, it discusses causes, symptoms, and diagnostic tests. It emphasizes that no single test can diagnose thyroid disease and that a combination of tests is needed. It also notes that subtle thyroid abnormalities may be missed by standard lab ranges and that patient symptoms should be considered.
ROLE OF LAB IN COMMON PEDIATRIC EMERGENCIESMoustafa Rezk
The document discusses several common pediatric emergencies and the role of the laboratory in evaluating and diagnosing them. It provides details on diabetic ketoacidosis (DKA), including causes, symptoms, differential diagnosis, and the 10 most important laboratory tests for evaluation. It also discusses dehydration, noting that volume depletion is more common than dehydration in children, and outlines different types of volume depletion based on sodium levels. Key laboratory tests for assessing dehydration include electrolytes, bicarbonate, potassium, and osmolarity.
The document discusses various pre-analytical and post-analytical errors that can occur in clinical laboratories. It notes that errors commonly occur in specimen receiving, sampling, transport, and results reporting. Some common errors include entering the wrong patient data, incomplete patient information, misidentifying tests, collecting samples from patients with the wrong test orders, and not fulfilling all requested investigations. The document also discusses various biological and environmental factors that can influence laboratory test results, such as patient posture, exercise, underlying medical conditions, drug use, and diet. Proper specimen collection and handling is important to avoid pre-analytical errors.
Oxidative stress occurs when there is an imbalance between free radicals and antioxidants in the body. Free radicals are reactive molecules that can damage cells, and are produced through normal metabolic processes or external factors like pollution. They react with lipids, DNA, and proteins which can lead to cell injury. Lipid peroxidation caused by free radicals can disrupt cell membranes and form toxic compounds. Oxidative stress is measured indirectly through biomarkers of damage like lipid peroxides and DNA oxidation products, or directly through assays of reactive oxygen species and antioxidants. Maintaining the balance between free radicals and antioxidants is important for health.
The document discusses kidney function and diseases. It describes the anatomy and functional unit of the kidney, the nephron. The kidneys perform crucial functions like regulating fluid balance and electrolytes, maintaining acid-base balance, and controlling blood pressure. Laboratory tests can assess glomerular function through measuring the glomerular filtration rate (GFR) using clearance tests of substances like creatinine and urea. Other tests evaluate tubular function and kidney handling of electrolytes and acids/bases. Diseases are discussed like chronic kidney disease and acute kidney failure where laboratory findings are altered.
Here is the updated list of Top Best Ayurvedic medicine for Gas and Indigestion and those are Gas-O-Go Syp for Dyspepsia | Lavizyme Syrup for Acidity | Yumzyme Hepatoprotective Capsules etc
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
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THE URINARY PROTEOMICS:A TOOL TODISCOVER NEW AND POTENT BIOMARKERS FORKIDNEY DAMAGE
1. THE URINARY PROTEOMICS:A TOOL TO
DISCOVER NEW AND POTENT
BIOMARKERS FOR
KIDNEY DAMAGE
ByBy
Prof. Moustafa Rizk
Prof. of Clinical Pathology
Faculty of Medicine, University of Alexandria
Wednesday 07/04/2010
05:04 AM1
3. What is proteomics?
Proteomics is the systematic study of proteomes,
which describes the entire protein content of
one or all cells of an organism as well as of
bodily fluids such as blood, urine and sweat.
While the genome of an organism is considered to be mostly static,
the proteome shows dynamic properties with protein profiles
changing depending on a variety of extra- and intracellular stimuli
(i.e. cell cycle, temperature, differentiation, stress, apoptotic signals)
05:04 AM3
4. TYPES OF PROTEOMICS
1-Expression proteomics, : This is the
qualitative and quantitative study of the
expression of total proteins under two different
conditions. For example, expression
proteomics of normal cells and diseased cells
can be compared to understand the protein that
is responsible for the diseased state or the
protein that is expressed due to disease.
3-Functional Proteomics:This is an
assembly type of proteomic method to
analyze and understand the properties of
macromolecular networks involved in the life
activities of a cell.
4-Clinical proteomics, a comparative approach of
normal and abnormal status of cells, tissues or bodily
fluids , is still a promising new analytic discipline with the
following main aims:
(i) discovery of biomarkers allowing early detection, risk
management or therapeutic monitoring of diseases for the
establishment of individualized treatment procedures.
(ii) identification of protein targets for the development of
new mechanistic intervention therapies with the promise of
an improved clinical outcome.
05:04 AM4
2-Structural Proteomics:Structural
proteomics, as the name indicates, is about
the structural aspects, including the three-
dimensional shape and structural
complexities, of functional proteins
5. So a major goal in the field of clinical proteomics
is to identify disease biomarkers in biological
fluids that can be measured relatively
inexpensively for early diagnosis of disease.
05:04 AM5
6. An important challenge in this process is to develop a
rational means of reducing the complexity of the
proteome of body-fluid samples to enhance the
detectability of relatively low-abundance proteins that
may have special pathophysiological significance.
05:04 AM6
7. Most of the focus thus far has been on
proteomics of blood (serum or plasma) .
Because urine can be collected noninvasively
in large amounts, it provides an attractive
alternative to blood plasma as a potential
source of disease biomarkers .
05:04 AM7
8. The increasing number of patients suffering from
chronic renal failure represents one of the
major challenges which the nephrologists are
facing worldwide.
For a better therapeutic outcome of this
disease, earlier detection is urgently warranted
in routine clinical practice.
05:04 AM8
9. Origin of urinary proteins and peptides :
1- Glomerular filtration
2- Tubular secretion
3- Epithelial cells shed from the kidney and
urinary tract
4- Secreted exosomes
5- Semen.
05:04 AM9
11. Urinary exosomes containing apical membrane
and intracellular fluid are normally secreted
into the urine from all nephron segments, and
may carry protein markers of renal dysfunction
and structural injury.
Derived from all cell types that face the urinary
space including glomerular podocytes, renal
tubule cells, and the cells lining the urinary
drainage system 05:04 AM11
12. Process of exosome formation and release into the urine
Hoorn, E. J., Pisitkun, T and Knepper, M. A. (2005) Prospects for urinary proteomics:
exosomes as a source of urinary biomarkers. Nephrology (Carlton) 10, 283–290
1- Apical membrane proteins
undergo endocytosis followed
by targeting to the
MVB.
2- The membrane proteins are
segregated initially in the MVB
outer membrane and then are
internalized by membrane
invagination, encapsulating
cytosolic proteins in the process.
3- After accumulation of
numerous internal vesicles, the
outer membrane of the MVB
fuses with the apical plasma
membrane releasing its internal
vesicles, called exosomes, into
the urinary space.
4- Exosomes contain both membrane and cytosolic proteins.
Multivesicular bodies
05:04 AM12
13. Urine sample collection, storage, and processing
procedures for isolation of urinary exosomes
Sample collection (protease inhibitor) solution (volume per 50 ml urine)
1.67 ml of 100 mM sodium azide (NaN3)
2.5 ml PMSF (2 mg/ml in isopropyl alcohol, stable 4°C for several months)
50 µl Leupeptin (1 mg/ml in ddH2O, stable 1 week at 4°C, 6 months at -20°C)
Collection
If possible, determine the collection time and volume.
Collect the first or second morning urine, 10~100 ml .
First morning urine: first urine after waking (no fluid or fruits after 9 PM the prior evening).
Second morning urine: Discard the first urine. Collect the next voided urine. May have breakfast and undergo regular
activity between first and second morning urine.
Can also use random urine samples
Storage
Add sample collection (protease inhibitor) solution
If possible, process samples immediately (refrigerate at 4 °C).
If the urine samples cannot be processed immediately, the samples should be frozen at -80 °C (NOT -20 °C).
50 ml of urine samples are aliquoted in 50 ml tubes and stored at -80°C as soon as possible after collection.
Defrosting and processing
Frozen urine samples should be thawed at room temperature (requires 3 hrs for a 50 ml urine sample). Avoid prolonged
thawing on ice (4 °C).
While urine is defrosting (i.e., is still a mixture of ice and water), extensively and vigorously vortex for one minute.
After sample completely thaws, vigorously vortex for an additional 30 sec, then proceed to differential centrifugation for
urinary exosome isolation.
Insufficient vortexing will result in major loss of urinary exosomes.
05:04 AM13
14. Classifications of exosomal identified proteins
from renal epithelia
1 - Glomerular podocytes (podocin and podocalyxin)
2 - Proximal tubule (e.g., megalin, cubilin, APN, AQP1)
3 - Ascending limb of Henle (e.g., THP, CD9)
4 - Distal convoluted tubule (e.g., NCC),
5 - Collecting duct (e.g., AQP2, mucin-1, and the Rh type C )
6 - Transitional epithelium of the urinary bladder ( uroplakin-1 and -2)
Thus, proteomic analysis of urine can potentially provide insight into the
physiological or pathophysiological processes in every epithelial cell type
facing the urinary space.
05:04 AM14
15. Naturally occurring human urinary peptides and proteins
Establishment of a high resolution proteome database ranging
from 800–17,000 Da from over 3,600 individual samples
using capillary electrophoresis coupled to mass
spectrometry, yielding an average of 1,500 peptides per
sample.
All processed data were deposited in an SQL database,
currently containing 5,010 relevant unique urinary peptides
that serve as classifiers for diagnosis and monitoring of
diseases, including kidney and vascular diseases.
Of these, 352 have been sequenced to date.
Nature Precedings : hdl:10101/npre.2007.1219.1 : Posted 11 Oct 2007 05:04 AM15
16. Nature Precedings : hdl:10101/npre.2007.1219.1 : Posted 11 Oct 2007
CE-MS data from 28 different pre-selected pathophysiological conditions
05:04 AM16
17. Distribution of native peptides identified with respect to their protein precursor (described
by SwissProt protein name and gene symbol). Comparison of peptides to other references)
A database of naturally occurring human urinary peptides and proteins for use in clinical applications
Nature Precedings : hdl:10101/npre.2007.1219.1 : Posted 11 Oct 2007
05:04 AM17
20. Proteomics is increasingly used in the discovery of
renal biomarkers mostly in:
1 - Cancer screening
2 - Glomerular diseases in native kidneys
3 - Acute rejection
4 - BK virus nephropathy as a complication in kidney
transplants
Early detection of graft rejection
Specific new biomarker candidates for other renal disease
detected by proteomic analysis
5- Predict the need for operation in newborns presenting
with unilateral uretero-pelvic junction obstruction05:04 AM20
21. Cancer screening
1- Renal cell carcinoma (RCC) is the sixth leading
cause of cancer death and is responsible for 11,000
deaths per year in the US.
2- In this study, a comprehensive proteomic analysis
to identify biological processes involved in clear cell
RCC (ccRCC).
3- Urinary markers of RCC were investigated
which could be applied to high-risk patients, or to
those being followed for recurrence, for early
diagnosis and treatment, thereby substantially
reducing mortality of this disease.
Pathway analysis of kidney cancer using proteomics and metabolic profiling.Bertrand Perroud, et
al Mol Cancer. 2006; 5: 64. 05:04 AM21
22. Using 2-dimensional electrophoresis and mass
spectrometric analysis, 31 proteins were identified which
were differentially expressed with a high degree of
significance in ccRCC as compared to adjacent non-
malignant tissue, and some of these were confirmed by
immunoblotting, immunohistochemistry, and comparison
to published transcriptomic data.
Pathway analysis of kidney cancer using proteomics and metabolic profiling.Bertrand Perroud, et al Mol
Cancer. 2006; 5: 64.
BlueBlue : Underexpression
BlackBlack: Equal expression
RedRed : Overexpression
05:04 AM22
23. A highthroughput proteomic approach was applied to the evaluation of
urine samples from type 2 diabetic patients and identified a protein
profile that accurately predicted nephropathy in advance of an
increase in albuminuria. These results warrant further studies to
determine the applicability of this approach within other populations,
and, more specifically, within other larger cohorts of diabetic patients
with prospectively collected samples.
Finally,further characterization and evaluation of the proteins in the
biomarker profile are needed to demonstrate whether they are
biologically important in the development of nephropathy.
Prediction of Diabetic Nephropathy Using Urine Proteomic Profiling 10 Years Prior to
Development of Nephropathy.Diabetes Care 30:638–643, 2007
05:04 AM23
25. Detection of Acute Tubulointerstitial Rejection by Proteomic
Analysis of Urinary Samples in Renal Transplant Recipients
A distinct urinary polypeptide pattern identified 16 out
of 17 cases of acute tubolointerstitial rejection, but
was absent in two cases of vascular rejection. Urinary
tract infection resulted in a different polypeptide
pattern that allowed to differentiate between infection
and acute rejection in all cases..
Detection of acute rejection by CE-MS offers a
promising non-invasive tool for the surveillance of
renal allograft recipients..
05:04 AM25
26. Copyright restrictions may apply.
Mosley, K. et al. Rheumatology 2006 45:1497-1504; doi:10.1093/rheumatology/kel351
Candidate biomarker discovery
Urine samples from inactive (n= 49) and active (n= 26) lupus nephritis patients
05:04 AM26
27. Gel based and gel free proteomics methods in urinary
proteome analyses:
05:04 AM27
29. Several methods have been used to search for biomarkers
in urine samples :
1- Two-dimensional electrophoresis
2- Surface-enhanced laser desorption/ionization
time- of-flight (SELDI-TOF)
3- Capillary electrophoresis.
4- Matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) to identify -β2
microglobulin (B2M) in the urine as a
potential biomarker in acute rejection for
kidney transplant recipients.
5- Another method recently introduced is iTRAQ.
(Multiplexed Isobaric Tagging Technology)
05:04 AM29
37. MALDI characteristics
1. Soft ionization method, so results predominantly in the
generation of singly charged ions hence the spectra are relatively
easy to interpret.
2. Provides molecular weight information.
3. MALDI deals well with thermolabile, non-volatile organic
compounds especially those of high molecular mass
4. MALDI is used successfully for the analysis of proteins,
peptides, glycoproteins, oligosaccharides, and oligonucleotides..
05:04 AM37
38. Types of Mass Analyzers
• Sector Mass Analyzers
• Time Of Flight (TOF)
• Quadrupole
• Quadrupole Ion Trap
• Ion Cyclotron Resonance
05:04 AM38
48. What is a mass spectrum?
Mass spectrum: graphic
representation of ions
separated according to their
m/z ratio.
It is a plot of relative
abundance versus mass-to-
charge ratio (mz).
05:04 AM48
50. Summary of the proteomic platforms used for urine
analysis, their advantages and disadvantages.
05:04 AM50
51. Conclusion
1- The adequate diagnosis of complex diseases e.g., renal
disease with a single biomarker seems to be an illusion.
2- A Multiple biomarker assay could deliver a better and a
more individualized diagnosis and allow therapeutic
strategies that delay or prevent the progression of the
disease.
3- Due to many limitations and uncertainties,
urinary proteomics at present cannot replace invasive
standardized diagnostic procedures such as the renal
biopsy, but holds great promise and potential for future
highly improved diagnosis and care of the patient in
nephrology.
05:04 AM51
53. Copyright restrictions may apply.
Thongboonkerd, V. Nephrol. Dial. Transplant. 2010 25:11-16; doi:10.1093/ndt/gfp476
Schematic summary of methodologies and applications of renal and urinary proteomics
05:04 AM53
Editor's Notes
DNA Basics.. Inside the nucleus, most of the DNA exists as a double helix.. The genetic information is stored in the sequences of nucleotide bases (A, T, G or C) that form the rungs of the ladder. Each rung is formed by a pair of nucleotide bases touching each other, one base attached to one strand backbone, and the other attached to the other strand backbone. An "A" nucleotide always pairs with a "T," and a "G" always pairs with a "C." In order to synthesize a protein, the cell reads the genetic information of the gene for that protein by "transcribing" a molecule of RNA from the gene. For transcription, the strands of the DNA double helix must partially separate so that the bases that form RNA can assemble according to the rules of complementary basepairing.. The transcribed strand of RNA acts as a "messenger" that carries the genetic information from storage in the nucleus to the protein manufacturing modules in the cytoplasm. The protein folds into a functional three-dimensional structure that depends on the linear sequence of amino acids. Proteins are large biological molecules made of subunits called amino acids that are attached to one another in chains, like the cars of a train. There are twenty different kinds of amino acids used in proteins, and most proteins contain hundreds of these subunits. Each protein has a specific number and sequence of amino acids, and this sequence determines what properties that protein will have. In order for a cell to synthesize a specific protein, it must access an "information bank" in which amino acid sequences are stored; this information bank is comprised of the organism's genes, which contain the amino acid sequences encoded in molecules of deoxyribonucleic acid (DNA). The identification of these targets should be carried out at the genomic, transcriptomic, proteomic and metabolomic level.
What is Proteomics?
In contrast, proteomics focuses on the identification, localization, and functional analysis of the protein make-up of the cell. The proteins present in a cell, together with their function, sub-cellular location, and perhaps even structure, change dramatically with the organism, and the conditions faced by their host cells including: age, checkpoint in the cell cycle, and external or internal signaling events.
Thus, there are many proteomes for each organism and consequently, the quantity and complexity of the data derived from the sequencing and mapping of the human proteome are estimated to be at least three times greater than that involved in the human genome project. Acquiring, analyzing, and interpreting these vast data sets requires a series of well-integrated, high-throughput technologies to lead the researcher from experimental design to biological insight.
The field of proteomics is particularly important because most diseases are manifested at the level of protein activity. Consequently, proteomics seeks to correlate directly the involvement of specific proteins, protein complexes and their modification status in a given disease state.Such knowledge will provide a fast track to commercialization and will speed up the identification of new drug targets that can be used to diagnose and treat diseases.
Expression Proteomics: This is the qualitative and quantitative study of the expression of total proteins under two different conditions. For example, expression proteomics of normal cells and diseased cells can be compared to understand the protein that is responsible for the diseased state or the protein that is expressed due to disease. Using this method disease-specific protein can be identified and characterized by comparing the protein-expression profile of the entire proteome or of the subproteome between the two samples.
For example, tumor tissue samples from a cancer patient and the same type of tissue from a normal person can be analyzed for differential protein expression. Using two-dimensional gel electrophoresis, mass spectrometry combined with chromatography and microarray techniques, additional proteins that are expressed in the cancer tissues or the proteins, which are absent, or those, which are over expressed and under-expressed can be identified and characterized. Identification of these proteins will give valuable information about the molecular biology of tumor formation.
Structural Proteomics:Structural proteomics, as the name indicates, is about the structural aspects, including the three-dimensional shape and structural complexities, of functional proteins. This includes the structural prediction of a protein when its amino acid sequence is determined directly by sequencing or from the gene with a method called homology modeling. This can be carried out by doing a homology search and computational methods of protein structural studies and predictions.
Apart from this, structural proteomics can map out the structure and function of protein complexes present in a specific cellular organelle. It is possible to identify all the proteins present in a complex system such as ribosomes, membranes, or other cellular organelles and to characterize or predict all the proteins and protein interactions that can be possible between these proteins and protein complexes. Structural proteomics of a specific organelle or protein complex can give information regarding supra-molecular assemblies and their molecular architecture in cells, organelles, and in molecular complexes.
Functional Proteomics:This is an assembly type of proteomic method to analyze and understand the properties of macromolecular networks involved in the life activities of a cell. With these methods it will be possible to identify specific protein molecules and their role in individual metabolic activities and their contribution to the metabolic network that operates in the system. This forms one of the major objectives of functional proteomics. For example, the recent elucidation of the protein network involved in the functioning of a nuclear pore complex has led to the identification of novel proteins involved in the translocation of macromolecules between the cytoplasm and nucleus through these complex pores.
Functional proteomics is yielding large databases of interacting proteins, and extensive pathway maps of these interactions are being scored and deciphered by novel high-throughput technologies. However, traditional methods of screening have not been very successful in identifying protein-protein interactionsand their inhibitors. The identification and measurement of changes in the concentration of specific proteins that cells make as a result of their genetic response to specific toxicants, and how these proteins are related to each other and to the specific biological condition of the cell, also fall under functional proteomics.
Proteomics thus holds significant promise for the discovery of diagnostic or prognostic protein markers, for the detection of new therapeutic targets and as a powerful tool to further our understanding of basic biological processes and mechanisms. The subject, called targeted proteomics, therefore involves the integration of a number of technologies including the selective targeting of proteins with activities of interest, multi-step sample preparation, and mass spectrometry. Examining changes in these proteins within cells under different physiological conditions will offer insights into understanding cellular and molecular mechanisms that cannot currently be obtained through traditional biological studies that usually focus on the detailed analysis of individual biomolecules.
Profiling of these thousands of proteins and peptides (polypeptides) using high resolution capillary electrophoresis coupled mass spectrometry (CE-MS) allows better understanding and in depth diagnosis of most known diseases. We have established a proteomic database of human urinary polypeptides. This library can be used as a rich source of biomarkers for a wide range of diseases. More than 3,300 urine samples were analysed CE-MS, yielding an average of 1,500 polypeptides per sample. All these CE-MS data were processed to identify and quantify individual peptides. The resulting database currently contains 4,954 different urinary peptides that can be used to build robust classifier models for diagnosis, stratification, and monitoring of a wide range of diseases, such as prostate and bladder cancer as well as kidney and vascular diseases. Two-dimensional gel electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues or biological fluids. This technique permits separation and characterization of proteins according to their charge/ion strength and molecular weight, in two consecutive gel electrophoresis steps: proteins are first separated by isoelectric focussing, according to their isoelectric points and then distinguished according to their molecular weights in SDS-polyacrylamide gel electrophoresis. 2-D gel-electrophoresis is generally labour- and time-intensive and without strict standardization in the applied reagents, apparatus and software for the analysis usually not routinely applicable in clinical settings. Gel-free proteome analysisReduced sample requirement, high throughput and automation are also important conditions for the integration of proteomics in routine laboratories. For this reason, different methods have been developed, which effectively couple high-end mass spectrometry to array formats, to capillary electrophoresis or to chromatography. The surface-enhanced laser desorption/ionization (SELDI) technique offers such an opportunity for urine analysis. Small amounts of native urine samples can be applied to the surface of a SELDI ProteinChip without prior concentration or precipitation of the urinary proteins . The bound proteins may then be directly analysed by MALDI-TOF-MS . Also CE-MS coupled the high-resolution properties of capillary electrophoresis (CE) with the powerful identification ability of the electrospray time-of-flight MS to profile and sequence urinary proteins. Liquid chromatography coupled to mass spectrometry (LC-MS) also offers a gel-free alternative for sensitive urine proteome analysis. Thus, protein profiles or single identified proteins may be characterized as disease-specific protein pattern or biomarkers which, however, must be validated in controlled retro- and prospective clinical studies.
The use of urinary biomarkers to diagnose disease is a long-standing practice. Ancient clinicians detected glucose in the urine by tasting it or observing whether it attracted ants.The presence of albumin in the urine has been measured as an indicator of renal disease for centuries and in early times could be detected via the so-called “foam test” to determine whether albumin was present in the urine in large amounts. Even today clinicians frequently shake a urine sample to determine whether it develops a froth, prima facie evidence for a high level of protein, which often is indicative of glomerular disease. In that tradition, studies to identify biomarkers of disease in the urine have been an underlying component of investigative medicine throughout the 20th century and the early 21st century. These studies have been based on knowledge of the pathophysiology of disease to identify putative biomarkers that could be tested in clinical trials.
Origin of urine proteins: in the urinary system high molecular weight proteins (> 40 kDa) are hold back in the glomerular part, whereas the low molecular weight proteins are absorbed in tubulus. Glomerular proteinuria led to increased release of high molecular weight proteins and tubular proteinuria is characterized by high excretion of low molecular weight proteins.
Recently, exosomes containing both membrane and cytosolic proteins were isolated from normal human urine by differential centrifugation, and exosomal proteins related to kidney diseases were identified by LC-MS/MS . Exosomal protein biomarkers might be clinically useful biomarkers; Du Cheyron et al reported that NHE3, a membrane protein increased in acute tubule necrosis (ATN) patients but not in prerenal azotemia or acute glomerular diseases . We thought that this urinary compartment, containing a reduced protein complexity and depleted predominant proteins, might be used for biomarker discovery, particularly for proteins that might indicate structural renal disease.In present study, Fetuin-A was located in the cytoplasm of damaged proximal tubules that were attached on the basement membrane and at even higher concentration in proximal tubule cells that had detached from the tubule basement membrane. While exosomal Fetuin-A may be synthesized by the kidney, it may also appear in the urine as result of incomplete proximal tubule processing in proteinuria states (a form of overflow proteinuria) or released during tubular cell apoptosis.
Capillary electrophoresis coupled to mass spectrometry (CE-MS) enables reproducible and robust high-resolution analysis of several thousand low-molecular-weight urinary proteins/ peptides in less than an hour 1. This approach has been used to analyze urine samples from healthy volunteers and patients with various diseases. The high number of datasets analyzed using identical conditions on the same instruments allows the establishment of a low-molecular-weight proteome database that can serve as a basis for the diagnosis, classification, and monitoring of a wide range of diseases using proteome analysis. The data were evaluated using Mosaiques Visu26 resulting in a list of
peptides/proteins defined by mass, migration time, and ion-counts, serving as a measure of relative abundance. Different charge states of identical peptides/proteins were combined and included as a single identification in the database. A list of tentatively identified peptides of any sample is obtained and subsequently calibrated using “internal standards”, peptides generally present in urine. This allows the digital compilation of individual datasets into a specific “disease group” that can be compared to any desired “control group”, enabling the identification of statistically significant changes that result in the definition of potential biomarkers. Only datasets that fulfilled all quality control criteria were subsequently utilized and included in the database,which currently includes CE-MS data from 28 different pre-selected pathophysiological conditions .
All urine samples were prepared identically and analyzed by CE-MS analysis, using
identical instruments. Analysis resulted in individual data sets containing information on
generally 1,200-2,000 peptides and proteins per sample. All information recommended by
the “minimum information about proteomics experiments” (MIAPE) guidelines about
proteome analysis using CE and MS is recorded, and available upon request. The data
were evaluated using MosaiquesVisu , resulting in a list of
peptides/proteins defined by mass, migration time, and ion-counts, serving as a measure
of relative abundance. Different charge states of identical peptides/proteins were
combined and included as a single identification in the database. A list of tentatively
identified peptides of any sample is obtained and subsequently calibrated using “internal
standards”, peptides generally present in urine. This allows the digital
compilation of individual datasets into a specific “disease group” that can be compared to
any desired “control group”, enabling the identification of statistically significant changes
that result in the definition of potential biomarkers. Only datasets that fulfilled all quality
control criteria were subsequently utilized and included in the database, which currently includes CE-MS data from 28 different pre-selected pathophysiological conditions .
All detected peptides and proteins in the 3,687 human urine samples that passed
all quality control criteria (on average, 1,724 peptides/proteins were detected in each individual urine sample, ranging from 983 to 4,094) were deposited in a Microsoft SQL database and subsequently matched for further analysis and comparison of individual samples. This process resulted in the definition of 116,869 different peptides and proteins.
Each peptide was assigned a unique identification number (Protein ID). As described
Previously, several of these peptides appeared sporadically, being observed in only
one or a few samples. To eliminate such peptides of apparently low significance, only
those present in more than 20% of the urine samples in at least one group (samples from patients with same disease) were further investigated. This noise-filtering process reduced the number of peptides available for analysis significantly: applying these limits, 5,010 “relevant” different peptides characterized by molecular mass [Da] and normalized CE migration time [min] could be detected.
Table 2: Compendium of all patients and healthy controls of the human urinary proteome database, which were used for the establishment of training- and test-sets. Additionally, all sensitivities and specificities including their confidence interval are shown.
A), the low molecular weight range between 4500 and 7000 Da in 10 urine samples from each studied group (10 from DM-NP, 10 from DM-WNP, and 10 from WDM-NP patients and 10 from healthy controls) illustrated in gel view in the left panel. The mass peak with m/z 6188 differentiating DM-NP from the other groups is highlighted with the box. The right panel indicates the mass spectral view from the same mass peak. (B), gel view from the middle molecular weight range, 10 000–18 000. Boxes indicate the peak masses m/z 11 774 and 14 766 found to be differentially excreted in DM-NP patient urines.
Gel based and gel free proteomics methods in urinary proteome analyses: Gel based
urine analysis using 2D gel electrophoresis proteins will be separated according to their masses and
pIs. After in-gel enzymatic digestion of the proteins the tryptic product can be analyzed by mass
spectrometry. The identification can be performed by data bank search. Gel-free urinary proteome
analysis. ProteinChip coupled to MALDI-TOF-MS (SELDI-TOF-MS) technology. Different types of
ProteinChip surfaces are available. The chips are spotted with different chromatographic surfaces for
urine protein binding. Bound proteins are then ionized with mass spectrometry resulting in protein
profiles. CE-MS coupled the high-resolution properties of capillary electrophoresis (CE) and the
powerful identification ability of the electrospray time-of-flight MS to profile urinary proteins. The
resulting protein pattern can be used for diseases discrimination. Liquid chromatography coupled to
mass spectrometry (LC-MS) offers also a gel free alternative for urine proteome analysis. Dihazi et
al. (11)
During standard electrospray ionisation, the sample is dissolved in a polar, volatile solvent and pumped through a stainless steel capillary. A high voltage of 3 or 4 kV is applied to the tip of the capillary, and as a consequence of this strong electric field, the sample is dispersed into an aerosol of highly charged droplets, a process that is aided by a nebulising gas flowing around the outside of the capillary. This gas, usually nitrogen, helps to direct the spray emerging from the capillary tip towards the mass spectrometer.
The charged droplets diminish in size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying gas which passes across the front of the ionisation source. Eventually charged sample ions, free from solvent, are released from the droplets, some of which pass through a sampling cone or orifice into the analyser of the mass spectrometer.
This is the electrospray needle –This is the orifice plate, with the tiny one millimetre central hole through which the test sample passes. .
MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisation. The sample is pre-mixed with a highly absorbing matrix compound and dried on plate. The matrix transforms the laser energy into excitation energy for the sample, which leads to sample ionization. In this way energy transfer is efficient and in the mean time the sample molecules are spared excessive direct energy that may otherwise cause decomposition.
Sector analyzers uses an electromagnet to deflect ions that are flying through the mass analyzer. Deflection is dependent on their mass-to-charge ratios, deflecting the more charged & lighter ions more. Only ions of specific m/z ratio are capaple of reaching the detector ond are represented in the mass spectrum. Sector instruments have higher resolution and greater mass range than quadrupole instruments, but require larger vacuum pumps and often scan more slowly
Time-of-flight (TOF) analyzer uses an electric field to accelerate the ions in the fligt tube through the same potential, and then measures the time they take to reach the detector. If the particles all have the same charge, then their velocities will depend only on their masses. Lighter ions will reach the detector first. This requires that the starting time (the time at which the ions leave the ion source) is well defined. Therefore ions are either formed by pulsed ionization method (MALDI). Properties of ToF analyzers: - very simple by using fixed voltages and not
requiring a magnetic field
- drawback of poor mass resolution, less then 500
- very high transmission efficiencies,
Quadrupole mass analyzer consists of four parallel poles or rods. Mass sorting depends on ion motion resulting from simultaneously applied constant and radiofrequency potentials on the Quadrupole rods which can be set to pass only a selected m/z ratio. All other ions do not have a stable transit through quadrupole mass analyzer and will colloide with the rods, never reaching the detector. Analysis is accomplished by systemically changing the field strength thereby changing m/z transmitted through the analyzer. A quadrupole mass analyzer acts as a mass selective filter.
The quadrupole ion trap works on the same physical principles as the QMS, but the ions are trapped and sequentially ejected. Ions are trapped in a three-dimensional quadrupole ion storage device. For detection of the ions, the RF and DC potentials are gradually altered resulting in ejection of the ions in order of increasing m/z. (mass-selective ejection). This 'stream' of ions is focussed onto the detector of the instrument to produce the mass
Recently developed mass analyzer with special capabilities
- Sensitive
- Relatively inexpensive
- fast scan rates
Ions characterized by a specific mass-to-charge ratio and affected by a magnetic field move in a circular path at a given cyclotron frequency which is mass dependent. By applying a radio-frequency voltage at the same frequency as the cyclotron frequency, the ions absorb energy and are accelerated to larger cyclotron radius When the rf signal is terminated, the cyclotron radius remains at its larger state. This phenomenon provides the basis for ion cyclotron resonance mass spectrometry because ions having a different cyclotron frequency are not accelerated.
When the radio-frequency current is turned off, each packet of ions of a specific m/q induces an image current that is detected by a pair of electrodes in the analyzer cell. At this larger orbit, they come close to the electrodes to induce a signal The decay over time of the image current is transformed from the time domain into a frequency domain signal by a Fourier transform. FT-ICR having the highest recorded mass resolution of all mass spectrometers. - very expensive, typically for specialized research applications
ion beam hits a conversion dynode (made of semiconductor material)
- electrons released, accelerated to next dynode
- intensifying cascade of 106-108 fold
An electron multiplier (continuous dynode electron multiplier) is a vacuum-tube structure that multiplies incident charges. In a process called secondary emission, a single electron can, when bombarded on metal (or PbO coated surface) induce emission of roughly 1 to 3 electrons. If an electric potential is applied between this metal plate and yet another, the emitted electrons will accelerate to the next metal plate and induce secondary emission of still more electrons. This can be repeated a number of times, resulting in a large shower of electrons all collected by a metal anode.
The output of the mass spectrometer shows a plot of relative intensity vs the mass-to charge ratio (m/z). The most intense peak in the spectrum is termed the base peak and all others are reported relative to it's intensity. The peaks themselves are typically very sharp, and are often simply represented as vertical lines.
The process of fragmentation follows simple and predictable chemical pathways and the ions which are formed will reflect the most stable cations and radical cations which that molecule can form. The highest molecular weight peak observed in a spectrum will typically represent the parent molecule, minus an electron, and is termed the molecular ion (M+).
The m/z values of the ions are plotted against their intensities to show the number of components in the sample, the molecular mass of each component, and the relative abundance of the various components in the sample.
The following diagram displays the mass spectra of three simple gaseous compounds, carbon dioxide, propane and cyclopropane. The molecules of these compounds are similar in size, CO2 and propane both have the same mass of 44 amu, and cyclopropane has a mass of 42 amu. Even though these compounds are very similar in molecular mass, it is a simple matter to identify them from their individual mass spectra.