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Proteomics of body liquids as a source for potential  methods for medical diagnostics  and  mass spectrometry Prof. Dr. Ev...
Modern biological mass spectrometers are mainly ESI- TOF  Measuring time of ion flights in vacuum MALDI-TOF Orbitraps  Mea...
Ortho – ESI TOF Bruker, Thermo, Applied Biosystems,  Agilent, Waters…….
Orthogonal TOF Dodonov  1986 Reflectron  Mamyrin  1973 Electrospray Gall 1984
API Ion source  Linear Ion Trap  C-Trap Orbitrap differential pumping differential pumping The Thermo Scientific* LTQ Orbi...
The main goal of our research is to connect the level of protein expression with diseases or to find disease biomarkers.  ...
Protein enzym Mass analyses fragmentation Isolated peptide Masses of peptide fragments Search in database scoring Protein ...
Protein энзим анализ масс 1 Массы фрагментов пептидов Поиск в базе T ор- down method -   direct mass spectrometry of prote...
KETAAAKFERQYL K    ETAAAKFERQYL KE      TAAAKFERQYL KET     AAAKFERQYL KETA    AAKFERQYL KETAA   AKFERQYL KETAAA    KFERQY...
… -CHR – C(O) – NH – CHR’-… Polypeptide backbone fragmentation b y c z a x Collisionally Activated Dissociation (CAD) Elec...
ECD spectrum of 11+ ions from bovine ubiquitin
 
Problem of methods based on MS/MS identification <ul><li>Sensitivity lost  – informative are only MS/MS spectra,   whose i...
The other possibility in proteomics –  usage of high mass measurement  accuracy mass spectrometry
(From Alan Marshall NHMFL)
Ion cyclotron resonance mass spectrometer can  measure masses with sub ppm accuracy Linear ion trap IR laser Electron gun ...
Other mass spectrometers with high accuracy  of mass measurements are available now Orbitraps Q-TOFs …… . Mass accuracy  1...
At accuracy level of  1  ppm elementary composition of peptide with mass up to  600  Da  and  amino acid composition of pe...
. If we are using liquid chromatography (LC) or Capillary electrophoreses (CE) we have another tag  - LC retention time or...
LC reproducibility-Agilent 1100
Thus, there is a possibility in bottom-up approach to  proteomics is to create using MS/MS a database for  accurate mass t...
VGLQR   YVQLR   SLR Validated accurate mass tag ( SLTLGIEPVSPTSLR ) ... T GLYCESQTPR SLTLGIEPVSPTSLR VGLQRYVQLRSLR  ... … ...
FT ICR I.Boldin,  E.Nikolaev ASMS May 2010 Dynamicaly harmonized  FT ICR cell Pressure limited (practically unlimited  mas...
Reserpine, Resolving Power 22,000,000  without apodization, 180 s transient
BSA, 0.3mg/ml, 100scans accumulated, accumulation time in collision cell 50ms   (7 Tesla) M Hn+
22s R  = 1.3*10 6 BSA (65 kD) high resolution mode on 7 Tesla magnet R  = 0.9*10 6
Nb 3 Sn Coils NbTi Coils 21 Tesla FT-ICR Magnet Field Center to Flange 600 - 1100 mm 110 mm Bore Current Leads, Cryocooler...
FT MS ESI Q-TOF ESI TOF Lab Lab Clinic Accurate mass tag retention time approach
The most attractive is human plasma, which contains practically all proteins (around 20000 non modified forms) Human Prote...
Proteins in blood N. Leigh Anderson‡ and Norman G. Andersn Protein concentrations are different by  1 1   orders of magnit...
The main task is searching for protein biomarker of early stages of diseases
Alzheimer’s disease is a progressive brain disorder  of elderly people  that gradually destroys a person’s memory and abil...
tangles   Plaques   A β   – Amyloid  A   1-42,  Beta-amyloid peptide The main component of Alzheimer’s plaques  (1984) Se...
<ul><li>Why normal protein (monomeric Beta-amyloid)  aggregates to form pathogenic plaques? </li></ul><ul><li>What molecul...
Pro 19 Substitution by proline Abolishes fibril formation Met 35 (O) Oxidation may be Important for toxicity and/or oligom...
The goal is to develop mass spectrometric  methodology to distinguish peptides containing  different isomeric forms of ind...
f ECD of  1-16 А β z10 z10 z9 z9 Z10 -57 (C α -C β  bond destruction) c9 c9 y9 C A β  1-16 (isoAsp 7 ) A β  1-16  (Asp 7 )...
Distinguishing aspartate/iso-aspartate in A β   – Amyloid by CID Y10 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z 0 10...
Quantitative analyses
z10-57 z10 c10 z9 c9 c8 ECD   mass spectra of six  Аβ1-16(α- Asp )  and  Аβ1-16(β- Asp )  peptide mixtures with different ...
Our recent results on detection of A β 1-16,  extracted from human blood
Prototype of molecular diagnostics method <ul><li>Extraction of A β  fraction (~ 500 ng) from 5 ml of human plasma </li></...
Body liquids available noninvasively Exhaled breath condensate Urine Saliva Tear Sweat
Noninvasive diagnostic of human breath system by mass spectrometry monitoring of exhaled breath condensate
Breath condenser ECoScreen Jaeger from  VIASYSHealthcare (Germany)
KERATINS  in individual EBC of young healthy nonsmoking donors
Other proteins in individual EBC of young healthy nonsmoking donors
Proteins overexpressed in samples of patients with COPD (Chronic obstructive pulmonary disease) and pneumonia COPD  ( n = ...
Monitoring of exhaled protein composition after human lung transplantation Before surgery  ( artificial lung ventilation )...
Analyses of urine proteom Sick Healthy Urine is available in large quantities  –  ideal analyte for noninvasive diagnostic...
<ul><li>Three fractions of voided urine is under investigation </li></ul><ul><li>Proteome (masses of 3-60 kDa) </li></ul><...
Before use some proteins as biomarker we need  to know its temporal variability and polymorphism (how different is its con...
Two kinds of sample donors People “from street” (blood donation center) and people in “special conditions”.
For “people from street” Decision to include a person to the study group Current control for urogenital and other patholog...
For “healthy people data base” subset we need urine samples from persons under well  controlled diet and having healthy li...
Those  are people p articipating In long term isolation experiments in the frame of space research  programs.  April- July...
Ground based experimental facility
 
April- July 2009
Sample concentration  Amicon Ultra Ultracel-15 3 k  Desalting and major protein removal Urine collection   Centrifugation ...
Database:  IPI.Human v.3.52 Parent Tolerance: ± 5.0 PPM (Monoisotopic) Fragment Tolerance: ± 0.50 Da (Monoisotopic) Fixed ...
What is in the DB ( Structured Query Language database) <ul><li>Run, in which this peptide was identified </li></ul><ul><l...
Our statistics of the collected AMT tags in the  long term isolation experiment 447  LC-MS (liquid chromatography coupled ...
Current statistics of urinary proteome database for ordinary healthy people Smokers (41 sample) and non-smokers(46 samples...
Current statistics of urinary proteome database 233 LC-MS (liquid chromatography coupled with mass  spectrometry) runs tot...
Influence of life stile on urine proteome Smokers vs. non-smokers urine proteome
40% 35% Using all peptides Peptides Proteins Non-smokers 2527 762 Smokers 1893 627 Total 2758 840 Peptides Proteins 78 549...
20% 21% Using all peptides Peptides Proteins Odd 2232 445 Even 2306 467 Non-smokers 2535 506 Peptides Proteins 61 406 49 3...
Peptides Proteins 25% 25% Using all peptides Peptides Proteins Selection1 1723 365 Selection2 1588 337 Smokers 1894 400 35...
! ! ! ! ! ! Differences in the numbers of observed proteins participating in particular biological process in urine of smo...
This type of proteome analyses should be  personalized !!
Quantitative analyses by  18 O labeling 25  Individual non-labeled samples Pool of labeled Pool of non- labeled 25 Individ...
C: RT: 69.40 - 87.80 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 ...
A List of Candidate Cancer Biomarkers for Targeted Proteomics Malu Polanski and N. Leigh Anderson Biomark Insights. 2006; ...
Our partners
Molecular & Cellular Proteomics 9:2424–2437,  2010. Prof. Harald Mischak  Mosaiques Diagnostics GmbH, Mellendorfer Strasse...
ROC curves for classification of patient cohorts with “CKD pattern.” ROC analysis for CKD diagnosis of the training set an...
Samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in a...
HPLC-MS run duration is about 1.5-2 hours UPLC-MS duration is about 10-15 minutes We need faster technology!!
<ul><li>Efficient ion accumulation prior to IMS </li></ul><ul><li>High mass accuracy, high dynamic range data acquisition ...
Mobility Drift time Thermal diffusion-limited maximum resolution Temporal spread ION MOBILITY SPECTROMETRY (IMS) T k densi...
ADDITIONAL ANALYTICAL PEAK CAPACITY DUE TO IMS Only 3 features discerned without drift time dimension ( * )
<ul><li>Conclusions </li></ul><ul><li>Accurate mass tag/retention time databases for human urine proteome and peptidome we...
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Evgeny nikolaev proteomics of body liquids as a source for potential methods for medical diagnostics and mass spectrometry

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  • Investigation of EBC of seventeen healthy non-smoking donors between 20 and 36 years of age revealed that the major proteins are cell keratins, whose spectrum, however, is polymorphous for different people. Pairs of cytoskeletal keratins 1/10 and 2/9 are invariant for mostly probes. No mutations in the sequences of these proteins in healthy donors have been detected. At the same time, other keratins are substantially different for individual sample.
  • Apart from keratins, dermcidin (known as a protein antibiotic originating in the sweat glands), prostaglandin H2 D-isomerase (PGDS2), alpha-1-microglobulin/bikunin precursor (AMBP), ubiquitin and cystatin A occurred also frequently (  30 % of donors). In the same time, some proteins appeared only in single instance. There were immunoglobulin light chain region, human basement membrane heparan sulfate proteoglycan core protein (HSPG2), leukocyte-associated immunoglobulin-like receptor 1 isoform a precursor (LAIR1), lysosomal membrane glycoprotein-2 (LAMP2), cerebroside sulfate activator (CSA), kininogen 1, serum albumin.
  • We found keratins in most of the samples of patients. These keratins were identified as “normal”, because they were detected in EBC of healthy donors. Additionally, specific peptides of keratins 3, 4, 8 were identified in COPD samples. These keratins were not found in healthy samples; therefore, they were named “abnormal”. It is worth noting that the keratin set identified in samples from patients with acute pneumonia was more varied. Keratins 4/13, 7/19, 8/18 and 15 were also identified in those samples. Peptides of certain other proteins uncharacteristic of healthy EBC samples were discovered in COPD and pneumonia EBC samples: namely, Junction plakoglobin, Desmoplakin, Dermokine, alpha-2-glycoprotein 1, Alpha-1-acid glycoprotein 2, Filaggrin-2, Dynein, Lysozyme, Collagen alpha-1(XVIII), Hornerin.
  • Результаты анализа белкового состава конденсата выдыхаемого воздуха согласуются с результатами клинического наблюдения пациента, перенесшего трансплантацию легких, и, таким образом, характеризуют его состояние.
  • Transcript of "Evgeny nikolaev proteomics of body liquids as a source for potential methods for medical diagnostics and mass spectrometry"

    1. 1. Proteomics of body liquids as a source for potential methods for medical diagnostics and mass spectrometry Prof. Dr. Evgeny Nikolaev Institute for Energy Problems of Chemical Physics and Institute for biochemical physics Rus. Acad. Sci., Moscow, Russia.
    2. 2. Modern biological mass spectrometers are mainly ESI- TOF Measuring time of ion flights in vacuum MALDI-TOF Orbitraps Measuring frequencies of ion oscillations Ion traps Measuring ion motion stability parameters FT ICR Measuring frequencies of ion oscillations in magnetic field
    3. 3. Ortho – ESI TOF Bruker, Thermo, Applied Biosystems, Agilent, Waters…….
    4. 4. Orthogonal TOF Dodonov 1986 Reflectron Mamyrin 1973 Electrospray Gall 1984
    5. 5. API Ion source Linear Ion Trap C-Trap Orbitrap differential pumping differential pumping The Thermo Scientific* LTQ Orbitrap XL* hybrid FTMS Alexander Makarov Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis. Anal. Chem. 2000; 72: 1156.
    6. 6. The main goal of our research is to connect the level of protein expression with diseases or to find disease biomarkers. Our Project:
    7. 7. Protein enzym Mass analyses fragmentation Isolated peptide Masses of peptide fragments Search in database scoring Protein and DND sequence database Mass analyses High throughput proteome analyses by tandem mass spectrometry methods Bottom-up method
    8. 8. Protein энзим анализ масс 1 Массы фрагментов пептидов Поиск в базе T ор- down method - direct mass spectrometry of proteins and peptides Ion transportation Mass analyses Masses of peptide fragments Search in database scoring Protein and DND sequence database fragmentation
    9. 9. KETAAAKFERQYL K ETAAAKFERQYL KE TAAAKFERQYL KET AAAKFERQYL KETA AAKFERQYL KETAA AKFERQYL KETAAA KFERQYL KETAAAK FERQYL KETAAAKF ERQYL KETAAAKFE RQYL KETAAAKFER QYL KETAAAKFERQ YL KETAAAKFERQY L Sequencing by MS/MS For unambiguous sequencing all peptide bonds should be broken
    10. 10. … -CHR – C(O) – NH – CHR’-… Polypeptide backbone fragmentation b y c z a x Collisionally Activated Dissociation (CAD) Electron Capture Dissociation (ECD) 1960s, 1990s 1998 Electron Detachment Dissociation (EDD) Electron Transfer Dissociation (ETD) 2004 2004 Infrared Multiphoton Dissociation (IRMPD) 1960s, 1995 157 nm UV Photodissociation Metastable-atom Induced Dissociation (MAID) 2004 2005
    11. 11. ECD spectrum of 11+ ions from bovine ubiquitin
    12. 13. Problem of methods based on MS/MS identification <ul><li>Sensitivity lost – informative are only MS/MS spectra, whose intensity is at least ~ 10 -fold lower than intensity of MS spectra </li></ul><ul><li>There is no possibility to detect all peptides in one run </li></ul><ul><li>Extra time for fragment spectra measurements causes longer chromatography time (application of UPLC is questionable for some types of MS instruments) </li></ul>
    13. 14. The other possibility in proteomics – usage of high mass measurement accuracy mass spectrometry
    14. 15. (From Alan Marshall NHMFL)
    15. 16. Ion cyclotron resonance mass spectrometer can measure masses with sub ppm accuracy Linear ion trap IR laser Electron gun Magnet
    16. 17. Other mass spectrometers with high accuracy of mass measurements are available now Orbitraps Q-TOFs …… . Mass accuracy 1-2 ppm ( intern. calib .), 5 ppm ( extern . calib. ) Resolution 2 0 000 -60 000 FWHM Rate of mass spectra measurements >20 Hz BRUKER micrOTOF-QII
    17. 18. At accuracy level of 1 ppm elementary composition of peptide with mass up to 600 Da and amino acid composition of peptide with mass up to 5 00 Da could be determined almost unambiguously It is not enough for peptide identification!
    18. 19. . If we are using liquid chromatography (LC) or Capillary electrophoreses (CE) we have another tag - LC retention time or CE retention time Accurate mass tag together with retention time Can identify peptide practically unambiguously! Accurate mass tag retention time Dick Smith group (PNNL)
    19. 20. LC reproducibility-Agilent 1100
    20. 21. Thus, there is a possibility in bottom-up approach to proteomics is to create using MS/MS a database for accurate mass tags and retention times as a reference base for fast quantitative measurements of proteins and peptides concentration in a sample
    21. 22. VGLQR YVQLR SLR Validated accurate mass tag ( SLTLGIEPVSPTSLR ) ... T GLYCESQTPR SLTLGIEPVSPTSLR VGLQRYVQLRSLR ... … T GLYCESQTPR SLTLGIEPVSPTSLR trypsinolyses Fragment (463-477) from Vasorin identification validation Vasorin (Homo Sapiens protein) 450 500 550 600 650 m/z 522.5 525.0 m/z LC- FTICR Accurate measured mass: 1568.8768 Putative mass tag from Homo Sapiens : SLTLGIEPVSPTSLR Calculated mass (1568.8773) And measured retention time 200 600 1,000 1,400 1,800 m/z y9 y8 b10 y7 b9 b8 y6 y12 y10 y11 b12 b6 y5 b7 b11 y13 b14 b13 y4 LC-MS/MS (e.g. with ion trap)
    22. 23. FT ICR I.Boldin, E.Nikolaev ASMS May 2010 Dynamicaly harmonized FT ICR cell Pressure limited (practically unlimited mass resolution)
    23. 24. Reserpine, Resolving Power 22,000,000 without apodization, 180 s transient
    24. 25. BSA, 0.3mg/ml, 100scans accumulated, accumulation time in collision cell 50ms (7 Tesla) M Hn+
    25. 26. 22s R = 1.3*10 6 BSA (65 kD) high resolution mode on 7 Tesla magnet R = 0.9*10 6
    26. 27. Nb 3 Sn Coils NbTi Coils 21 Tesla FT-ICR Magnet Field Center to Flange 600 - 1100 mm 110 mm Bore Current Leads, Cryocooler, and Quench relief for Zero-Loss 2.2 ° K Cryostat D. Markiewicz, NHMFL T. Painter, NHMFL J. Miller, NHMFL Y. S. Choi, KBSI Slide from Alan Marshall
    27. 28. FT MS ESI Q-TOF ESI TOF Lab Lab Clinic Accurate mass tag retention time approach
    28. 29. The most attractive is human plasma, which contains practically all proteins (around 20000 non modified forms) Human Proteome Detection and Quantitation Project:hPDQ N. Leigh Anderson, Norman G. Anderson, Terry W. Pearson, Christoph H.Borchers, Amanda G. Paulovich, Scott D. Patterson, Michael Gillette, Ruedi Aebersold and Steven A. Carr Mol Cell Proteomics Jan.2009
    29. 30. Proteins in blood N. Leigh Anderson‡ and Norman G. Andersn Protein concentrations are different by 1 1 orders of magnitude!!! There is no method to solve this analytical problem !
    30. 31. The main task is searching for protein biomarker of early stages of diseases
    31. 32. Alzheimer’s disease is a progressive brain disorder of elderly people that gradually destroys a person’s memory and ability to learn, reason, make judgments, communicate and carry out daily activities. Alois Alzheimer (1864-1915) 1906 - 2006 Alzheimer disease
    32. 33. tangles Plaques A β – Amyloid A  1-42, Beta-amyloid peptide The main component of Alzheimer’s plaques (1984) Sequenced in 1987 1 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 42 Anomalous accumulation of Beta-amyloid in the form of polymeric aggregates (plaques, tangles) causes Alzheimer
    33. 34. <ul><li>Why normal protein (monomeric Beta-amyloid) aggregates to form pathogenic plaques? </li></ul><ul><li>What molecular event is triggering this process? </li></ul><ul><li>Which part of the molecule is subjected to changing? </li></ul><ul><li>How to detect these changes? </li></ul>Questions to answer
    34. 35. Pro 19 Substitution by proline Abolishes fibril formation Met 35 (O) Oxidation may be Important for toxicity and/or oligomerization Asp 7 Isomerized by 75% In plaques Essential residues for self-association Primary structure elements controlling A β oligomerization
    35. 36. The goal is to develop mass spectrometric methodology to distinguish peptides containing different isomeric forms of individual amino acids and to apply this methodology to fragments of Alzheimer disease Beta-amyloid
    36. 37. f ECD of 1-16 А β z10 z10 z9 z9 Z10 -57 (C α -C β bond destruction) c9 c9 y9 C A β 1-16 (isoAsp 7 ) A β 1-16 (Asp 7 ) Distinguishing aspartate/iso-aspartate in A β – Amyloid by ECD
    37. 38. Distinguishing aspartate/iso-aspartate in A β – Amyloid by CID Y10 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1198.45 1585.55 1349.45 1448.55 1722.73 1585.55 1349.45 1448.45 1220.27 1722.45 m/z B6 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 798.36 819.91 756.36 811.36 861.82 847.91 793.36 819.91 861.91 847.91 811.36 793.36 756.27 Iso-aspartate aspartate aspartate 776.27 Y6 - H2O Y6 - H2O Y6 B11 B12 B13 B14 B10 DAEFRH D SGYEVHHQK b6 y10 CID Iso-aspartate B6 + H 2 O
    38. 39. Quantitative analyses
    39. 40. z10-57 z10 c10 z9 c9 c8 ECD mass spectra of six Аβ1-16(α- Asp ) and Аβ1-16(β- Asp ) peptide mixtures with different relative concentrations
    40. 41. Our recent results on detection of A β 1-16, extracted from human blood
    41. 42. Prototype of molecular diagnostics method <ul><li>Extraction of A β fraction (~ 500 ng) from 5 ml of human plasma </li></ul><ul><li>Preparation and extraction of A β 1-16 probe from A β pool </li></ul><ul><li>MS analysis of isoD7- to D7- A β 1-16 ratio in the probe </li></ul><ul><li>The next goal is to see this peptide in urine! </li></ul>
    42. 43. Body liquids available noninvasively Exhaled breath condensate Urine Saliva Tear Sweat
    43. 44. Noninvasive diagnostic of human breath system by mass spectrometry monitoring of exhaled breath condensate
    44. 45. Breath condenser ECoScreen Jaeger from VIASYSHealthcare (Germany)
    45. 46. KERATINS in individual EBC of young healthy nonsmoking donors
    46. 47. Other proteins in individual EBC of young healthy nonsmoking donors
    47. 48. Proteins overexpressed in samples of patients with COPD (Chronic obstructive pulmonary disease) and pneumonia COPD ( n = 17) pneumonia ( n = 13) <ul><li>Proteins of cytosceleton </li></ul><ul><li>Keratins 3, 4, 8 Keratins 4/13, 7/19, 8/18 and 15 </li></ul><ul><li>Junction plakoglobin Hornerin </li></ul><ul><li>Desmoplakin </li></ul>2. Proteases inhibitors Cystatin А Cystatin А Kininogen - 1 Kininogen -1 Cystatins В, М Alpha -1- antitrypsin 3. Other Osteopontin Cytoplasmic actin
    48. 49. Monitoring of exhaled protein composition after human lung transplantation Before surgery ( artificial lung ventilation ) 1 st month after surgery 15 months after surgery Pure protein spectrum because of disturbance of breath Dermcidin , Keratin 9 , Lysozyme , Ubiquitin Allograft adoptation and medical treatment Annexin 1 , Proteinases inhibitor, Bleomicine - hydrolase, keratin 8 Damaged epithelium removal Desmosomal proteins ( desmoglein , desmoplakin ) Epithelium healing Hornerin , filaggrin “ Normal” proteins Dermcidin , “normal” keratins , Cystatin A , Ubiquitin
    49. 50. Analyses of urine proteom Sick Healthy Urine is available in large quantities – ideal analyte for noninvasive diagnostic . Possibility of biomarker discovery is attracting big attention . 1500 proteins (from Mann’s group Adachi et al. Genome Biology 2006, V7, 9, R80) ; 2,362 proteins (Kentsis , A. et al. Proteomics Clinical Applications 2009, 3, (9), 1052-1061).
    50. 51. <ul><li>Three fractions of voided urine is under investigation </li></ul><ul><li>Proteome (masses of 3-60 kDa) </li></ul><ul><li>Peptidome (masses of 0.8-17 kDa) </li></ul><ul><li>Exosoms (50-90 nm vesicles secreted by a wide </li></ul><ul><li>range of cell types) </li></ul>
    51. 52. Before use some proteins as biomarker we need to know its temporal variability and polymorphism (how different is its concentration in body liquids of different individuals) To clarify this we need to investigate proteomes of hundreds of healthy individuals
    52. 53. Two kinds of sample donors People “from street” (blood donation center) and people in “special conditions”.
    53. 54. For “people from street” Decision to include a person to the study group Current control for urogenital and other pathology including kidney pathology, prostatitis, arterial hypertension, diabetes Analysis of archival information from medical records General blood analysis Examination of internist Blood pressure measurement Control for treatment with diuretics and excessive consumption of fluids
    54. 55. For “healthy people data base” subset we need urine samples from persons under well controlled diet and having healthy lifestyle? In this case we can test urine temporal variability and polymorphism
    55. 56. Those are people p articipating In long term isolation experiments in the frame of space research programs. April- July 2009. March 2010 + 500 days. (The Institute for medical & biological problems RAS)
    56. 57. Ground based experimental facility
    57. 59. April- July 2009
    58. 60. Sample concentration Amicon Ultra Ultracel-15 3 k Desalting and major protein removal Urine collection Centrifugation LC MS analyses Carboxymethylation and trypsinolyses
    59. 61. Database: IPI.Human v.3.52 Parent Tolerance: ± 5.0 PPM (Monoisotopic) Fragment Tolerance: ± 0.50 Da (Monoisotopic) Fixed Modifications: Carbamidomethyl (C) Variable Modifications: Oxidation(M) Digestion Enzyme: Trypsin Max Missed Cleavages: 2 Instrument type: Ion-trap Search engine: Mascot
    60. 62. What is in the DB ( Structured Query Language database) <ul><li>Run, in which this peptide was identified </li></ul><ul><li>Peptide sequence </li></ul><ul><li>What protein does this sequence belong to </li></ul><ul><li>Mascot score </li></ul><ul><li>Modifications </li></ul><ul><li>Measured mass </li></ul><ul><li>Theoretical mass </li></ul><ul><li>Measured charge </li></ul><ul><li>RT, when the peptide began to elute from the column </li></ul><ul><li>RT, when the peptide finished elution </li></ul>
    61. 63. Our statistics of the collected AMT tags in the long term isolation experiment 447 LC-MS (liquid chromatography coupled with mass spectrometry) runs totally: among them 25 samples from each of 6 volunteers have been collected during105 days of isolation experiment. The number of peptides in the database 3468 The number of urine proteins in the database 1055 443 core proteins (all patients have them in their urine)
    62. 64. Current statistics of urinary proteome database for ordinary healthy people Smokers (41 sample) and non-smokers(46 samples) Peptides Proteins Total 2758 840
    63. 65. Current statistics of urinary proteome database 233 LC-MS (liquid chromatography coupled with mass spectrometry) runs totally: 102 with samples from smokers, 131 with samples from non-smokers. Using all peptides Peptides Proteins Non-smokers 2527 762 Smokers 1893 627 Total 2758 840
    64. 66. Influence of life stile on urine proteome Smokers vs. non-smokers urine proteome
    65. 67. 40% 35% Using all peptides Peptides Proteins Non-smokers 2527 762 Smokers 1893 627 Total 2758 840 Peptides Proteins 78 549 213 231 1662 865
    66. 68. 20% 21% Using all peptides Peptides Proteins Odd 2232 445 Even 2306 467 Non-smokers 2535 506 Peptides Proteins 61 406 49 303 2003 229
    67. 69. Peptides Proteins 25% 25% Using all peptides Peptides Proteins Selection1 1723 365 Selection2 1588 337 Smokers 1894 400 35 302 63 306 1417 171
    68. 70. ! ! ! ! ! ! Differences in the numbers of observed proteins participating in particular biological process in urine of smokers and nonsmokers Transport, homophilic cell adhesion, lipid metabolic process, inflammatory response, innate immune response, epidermis development, defense response !
    69. 71. This type of proteome analyses should be personalized !!
    70. 72. Quantitative analyses by 18 O labeling 25 Individual non-labeled samples Pool of labeled Pool of non- labeled 25 Individual labeled samples MS 25 25
    71. 73. C: RT: 69.40 - 87.80 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 76.66 80.09 74.97 76.78 80.21 76.57 74.88 86.11 81.43 78.59 80.00 71.17 78.53 86.90 72.80 81.57 78.73 75.99 85.99 72.69 84.65 82.53 83.13 70.85 73.47 74.80 78.44 76.74 80.01 74.96 86.04 76.88 81.29 79.89 71.40 78.55 75.10 86.80 79.31 72.83 71.46 76.07 80.18 77.00 82.31 82.43 73.94 72.48 84.79 71.26 80.42 69.45 77.73 87.41 NL: 1.43E6 Base Peak MS 53_1-10_1ul NL: 1.36E6 Base Peak MS 53_o18_1- 10_1ul 574 575 576 577 578 579 580 581 582 m/z 0 20 40 60 80 100 0 20 40 60 80 100 575.31 z=2 575.81 z=2 576.32 z=2 577.32 z=2 577.82 z=2 578.32 z=2 NL: 1.43E6 53_1-10_1ul#5161 RT: 76.66 AV: 1 T: FTMS + p ESI Full ms [ 300.00-1600.00] NL: 1.34E6 53_o18_1- 10_1ul#5122 RT: 76.74 AV: 1 T: FTMS + p ESI Full ms [ 300.00-1600.00]
    72. 74. A List of Candidate Cancer Biomarkers for Targeted Proteomics Malu Polanski and N. Leigh Anderson Biomark Insights. 2006; 1: 1–48. The Plasma Proteome Institute list of 1261 proteins believed to be differentially expressed in human cancer As an initial approach, we have selected a subset of the candidates based on a set of criteria including number of total citations, number of recent citations, proportion of recent citations, known plasma concentration (implying existence of an assay) and clinical use in any context. This subset of 260 candidates 88 are detected in urine (Mann’s database) 75 (our database)
    73. 75. Our partners
    74. 76. Molecular & Cellular Proteomics 9:2424–2437, 2010. Prof. Harald Mischak Mosaiques Diagnostics GmbH, Mellendorfer Strasse 7–9, 30625 Hannover, Germany.
    75. 77. ROC curves for classification of patient cohorts with “CKD pattern.” ROC analysis for CKD diagnosis of the training set and the test set after unblinding is shown. 85.5% sensitivity and 100% specificity Peptide (800 to 17,000 Da) patterns distinguishing patients with CKD from HC 230 patients 379 healthy
    76. 78. Samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases.
    77. 79. HPLC-MS run duration is about 1.5-2 hours UPLC-MS duration is about 10-15 minutes We need faster technology!!
    78. 80. <ul><li>Efficient ion accumulation prior to IMS </li></ul><ul><li>High mass accuracy, high dynamic range data acquisition system </li></ul><ul><li>High IMS-TOF </li></ul><ul><li>sensitivity due to ion trapping and multiplexing </li></ul>ION MOBILITY SEPARATIONS IN HIGH THROUGHPUT ROTEOMICS: A NOVEL APROACH TO PROTEIN DETECTION AND IDENTIFICATION PERIMENTAL PLATFORM Mikhail Belov Biological Sciences Division Pacific Northwest National Laboratory
    79. 81. Mobility Drift time Thermal diffusion-limited maximum resolution Temporal spread ION MOBILITY SPECTROMETRY (IMS) T k density N Ze K b av   _ 2 16 3   K E L t drift  2 ln 16 T k LEZe R b d 
    80. 82. ADDITIONAL ANALYTICAL PEAK CAPACITY DUE TO IMS Only 3 features discerned without drift time dimension ( * )
    81. 83. <ul><li>Conclusions </li></ul><ul><li>Accurate mass tag/retention time databases for human urine proteome and peptidome were created </li></ul><ul><li>Subset of the database contains data from healthy people leaving in long term isolation conditions under the same diet </li></ul><ul><li>The new approach is developed for rapid analyses of urine proteome using AMT/RT database </li></ul><ul><li>Significant difference in protein and peptide content of urine of people with kidney diseases has been found inside some groups of proteins and peptides responsible for particular pathways </li></ul>
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