Clinical proteomics involves the large-scale study of proteins to better understand disease variability and identify new disease biomarkers and drug targets. There are two main approaches - hypothesis-based proteomics using protein microarrays, and discovery-based proteomics using gel-based or gel-free mass spectrometry techniques. Mass spectrometry allows identification of disease-associated changes in protein expression which can serve as biomarkers. Ideal biomarkers are sensitive, specific, accurate and cost-effective for diagnosing or monitoring diseases like Alzheimer's, cancer, and predicting disease progression or treatment response.

1. Clinical Proteomics
and Disease
Biomarkers
Presented By
MUGDHA RANGNATH
M.Sc. Biotechnology – 3rd
semester

2. introduction
 Proteome- the complete protein pool of an organism encoded by the
genome of a cell, tissue or organism.
 Proteomics- the large scale study of proteins, protein variations
and better understanding of the molecular basis of variability in
susceptibility to diseases that are associated with genetic diversity
and environmental factors.

3. Clinical proteomics
 the subset of proteomics activities in the field of medicine, which
promises to accelerate the discovery of new drugs targets and
protein disease markers useful for in vitro diagnostics.
study of proteins
and peptides
involved in pathological
processes
to develop new
diagnostic tests
to identify new
therapeutic targets

4. Methodologies
Two types of approaches are seen in clinical proteomics:-
1) Hypothesis based proteomics (Protein microarray).
2) Discovery based proteomics (Gel based and Gel free approach).

5. 1) Protein Microarray
 A protein microarray(protein chips) is high throughput method used
to track the interactions, activities of proteins and to determine
their functions.
 Protein microarray are miniaturized and parallel assay systems that
contain small amounts of purified proteins in a high density format.

6. 2) Mass Spectrometry (MS)
 Most widely used method in high
throughput proteomics study
 can measure the masses of
molecules such as peptides by
converting them into ions and
sorting them via electric field
according to their mass/charge
ratio
 Basic MS instruments are;
i. Ionization source- converts
the anlyte into gas phase ions
ii. Mass analyzer- separates ions
according to their mass
charge (m/z) ratio
iii. Ion detector- measures m/z
ratio.

8.  Discovery based proteomics contains (a) gel based MS
approach and (b) gel free MS approach
a) gel based:- 2D-Gel
electrophoresis
Separates proteins in pH gradient
according to isoelectric point
in first dimension and in
acrylamide matrix according to
molecular weight in second
dimension
Proteins are first of
all separated based on
their individual charges
in 1D(isoelectric
focusing).
Separation occurs in
2nd dimension when
proteins based on
difference in their size
is separated.

9.  Purification of
proteins: This step
involves extraction of
protein samples from
whole cell, tissue or sub
cellular organelles
followed by purification
using density gradient
centrifugation,
chromatographic
techniques (exclusion,
affinity etc.)
 Separation of proteins:
2D gel electrophoresis is
applied for separation of
proteins on the basis of
their isoelectric points in
one dimension and
molecular weight on the
other. Spots are detected
using fluorescent dyes.
 Identification of
proteins: The separated
protein spots on gel are
excised. The eluted
peptides are identified
using mass spectrometry.

10. b) Gel free approach
 MALDI (matrix assisted laser desorption ionization)
 Sample preparation is important for MALDI-TOF. Purify the protein sample
before going MALDI-TOF analysis. Because contaminants can disturb
incorporation of sample molecules with growing matrix crystals.
 Sample can mix in 1:2 ratio. Different types of matrix are used on sample
some of matrix are hydroxylated benzoic acid, cinnamic acid derivates.
 A good matrix should be able to absorb U.V wavelength of usually 237nm,
being easily excited and ability to transfer of proton to the sample molecules.
Main role of matrix is adsorption of energy from laser pulse, and then
transfer sample this energy can cause the vaporization of sample.
 Dried droplet technique is predominantly applied for MALDI-TOF analysis,
protein sample, mixed with matrix on a metal plate.

11.  In MALDI, samples containing peptides are embedded into specific
matrix molecule. The matrix absorbs the ionization laser beam and
transfers the energy into anlyte. Ionized peptides are directed via a
mass analyzer towards a detector, which generates MS Spectra with
each peak representing a m/z ratio ratio of ion.
Time-of-flight (TOF) analyzer is usually associated with MALDI ion
sources. It is one of the simplest mass analyzers. It measures the
mass:charge ratio of an ion by determining the time required for it to
transverse the length of flight tube to the detector.

12. SELDI (surface enhanced laser desorption ionization)
 SELDI-TOF MS is a combination of
chromatography and mass
spectrometry that uses an affinity
based method of mass spectrometry
comprising a protein chip modified
with a chromatographic affinity
surface.
 In SELDI, a sample is applied on
surface of a chip. The chip is then
placed in a vacuum chamber of the
SELDI where peptides and small
proteins are ionized and travel
towards a detector inversely
according to their masses.
 SELDI is mostly used in biomarker
discovery to compare protein levels in
serum samples from healthy and
diseased patients and has also applied
to the diagnosis of cancer and
neurological disorders.

15. biomarkers
 A biomarker is a measurable indicator of some biological state or
condition. They are used as analytical tools to early diagnosis of
disease. They are basically any kind of substances, structures, or
processes which could be measured in/outside the body.
According to the
definition given by
National Institutes of
Health Biomarkers in
1998, “Biomarkers are
evidence of biological,
pathogenic or
pharmacogenomic
response when
administered to any
therapeutic change”

16. Characteristics of an ideal biomarker
 Have great sensitivity, specificity,
accuracy, high predictive value
 Safe and easy to measure
 Cost efficient to follow up
 Modifiable with treatment
 Consistent across gender &
ethnic groups

17. Biomarkers in disease and diagnostic
processes
 Discovery of biomarkers constitute an essential part of biomedical research.
 The association of biomarkers to disease advances understanding of cellular and
molecular mechanisms of diseases since biomarkers can be direct causes of
diseases, secondary players in disease initiation and progression or mere signals
of pathological conditions.
 Molecular biomarkers can also play an important role in therapy as drug targets.
More specifically, the presence of molecular biomarkers in specific stages of
diseases will enable their use in disease diagnosis and prognosis.
 Blood, urine, cerebrospinal fluid provide the necessary biological information for
the diagnosis.

18. Biomarkers for Alzheimer’s Disease
Alzheimer’s Disease(AD) is one of the most common form
of dementia occurring in elderly population worldwide.
It is an age-dependent neurodegenerative disorder.
The first notable symptoms of AD include memory loss,
disorientation, and impairment of other cognitive functions.
Also it is associated with extracellular amyloid beta,
plaques deposition and accumulation of intracellular
neurofibrillary tangles composed of p-tau

19. The microtubule-associated protein(MAP), with six major isoforms, is
essential for the assembly and stability of the microtubules, an important
component of the neuronal cytoskeleton. In the AD brain, abnormally
hyperphosphorylated tau accumulate as neurofibrillary tangles. The
accumulation of dysfunctional amyloid beta tau are believed to mediate the
extensive loss of neuronsband synapses as well as the inflammatory
processes in the AD brain.

20. Presence of dementia is confirmed by analysing the Cerebrospinal fluid (CSF) with
established biomarker like amyloid beta protein, tau protein and phospho-tau
expression levels. CSF is known to act as a valuable source of biomarkers, since
besides in direct contact with the brain and spinal cord it provides a complete
representation of various biochemical and metabolic profiles of the brain.

21. Biomarkers in Oncology
Tumor metastasis is the process where spread of cancer from one organ to another
non-adjacent organ can cause death in patients. The major challenge in medicine to
describe the molecular and cellular mechanisms underlying tumor metastasis. Analyse
the protein expression correlated to metastatic process which help to understand the
mechanism and thus facilitate the development of strategies for the therapeutic
interventions and clinical management of cancer.

22. Some examples of Cancer
 Ovarian cancer
The glycoprotein antigen CA-125 is most
commonly measured tumor marker for
epithelial ovarian tumors which account
for 85-90% of ovarian cancers, and it is
used in the clinic to provide a prognosis,
monitor progression, and optimize the
care of women diagnosed with ovarian
cancer.
 Prostate cancer
PSA (prostate-specific antigen) is considered
the most important test for detecting,
staging, and monitoring early-stage
prostate cancer. Development of
biomarkers for the diagnosis, RNA-based
biomarkers and particularly protein
biomarkers. Human prostatic acid
phosphatase (PAP) and serum acid
phosphatase were reportedly the first
serum protein biomarkers for prostate
cancer.
 Breast cancer
High mobility group protein HMG-1/HMG
(HMGA1) abundance level was found to be
associated with breast cancer
clinicopathological features. Breast milk is
an appropaite cancer microenvironment
for identifying breast cancer biomarkers.
Besides milk, serum is also used for
identifying breast cancer-specific
biomarkers.
 Pancreatic cancer
Pancreatic cancer has been described as one
of the most lethal tumors. Carcinoma
antigen 19-9 (CA19-9) is a biomarker
which has been shown to be significant in
the diagnosis, prognosis, and management
of pancreatic ductal adenocarcinoma.

23. References
i. Wikipedia
ii.https://www.ncbi.nlm.nih.gov
iii.https://www.researchgate.net
iv.www.biologydiscussion.com
v. www.nptel.ac.in
vi.www.sciencedirect.com
vii.Google images.
Thank You!