Proteomics and its applications

Ravi Kumar, PhD

Proteomics

• The analysis of the entire protein complement in a given cell, tissue, body fluid and organism

• Proteomics assesses activities, modifications, localization, and interactions of proteins in complexes.

Genomics Transcriptome Proteomics

Introduction

• Proteome indicates the total proteins expressed by a genome in a cell or tissue

• Proteomics is increasingly being used to discover potential biomarkers noninvasively.

• Biomarkers detection might allow identification of patients who would benefit from further evaluation.

• With the development of proteomic techniques, proteome analysis provides a fast, non-invasive diagnostic tool for patients with various diseases.

• The advent of highly sensitive proteomic technologies can identify proteins associated with development of diseases well before any clinically identifiable alteration.

• MS has a high resolving power and identifies proteins with more accuracy

• Proteomic technologies can be applied for an un-biased examination to detect novel biomarkers that could play a critical role in disease diagnostics, treatment monitoring and prognosis.

History of proteomics• SDS-PAGE discovered by Laemmli in 1970.• O’Farrell (1975) applied IEF to protein samples

prior to SDS-PAGE to pioneer the concept of 2-DE.

• JJ Thomson (1913) identified M/Z ratio• Hillenkamp (1988) developed MALDI-MS. • Fenn (1988) developed ESI-MS• In 1993, Henzel et al. reported the first work

related to the identification of protein from the 2DE.

• Marc Wilkins coined the word ‘proteome’ in 1995 PROTEins complement of the genOMICS

2002 Nobel Prizes in Chemistry

Mass spectrometry for macromolecules

"for their development of soft desorption ionisation methods for MS analyses of biological macromolecules"

Koichi Tanaka John B. Fenn

Proteomics objectives

Protein/peptide separation

Identification and characterization of resolved proteins by MS

Data analysis and applications.

Experimental Work flow

Protein identification

(Sequest & Mascot)

Proper sample collection and storage

Sample pre-processing

Protein identification LC-MS/MS

Immuno depletion/protein concentration etc

In gel Tryptic digestion/ in-solution digestion

Bioinformatic Analysis

Protein separation by 1D SDS-PAGE/ 2DE

OrFractionation by LC

Proteomic techniques• Gel based

– SDS-PAGE– 2-DE

• Off gel base– LC (SCX, RP-LC, Immuno affinity)

• Quantitative proteomics– iTRAQ, ICAT, SILAC

• MS– MALDI, LC-MS, SLDI, CE-MS

Data bases

• Data analysis search engines• Sequest• MASCOT• X tandam• Peaks

• Protein data bases• NCBS• Swiss port

protein separation by SDS-PAGE

• SDS

• What is SDS-PAGE?

– SDS-PAGE a type of gel electrophoresis.

• What is the purpose of doing gel electrophoresis?

– It has been seen that by running a gel we are able to identify more proteins from the sample.

• An electric current is applied across the gel, causing proteins will differentially migrate based on their molecular mass.

• Staining will be done by using Coomassie Blue dye R-250, colloidal CBB G-250, Sypro ruby and silver stain

• periodic acid-Schiff (PAS), will be used to detect glycoproteins.

2-DE• Proteins separates based on• PI• MW

“You’ve got one protein missing …”

“No, you’ve one extra protein !”

LC

• RP-LC

• SCX

• Immuno affinity

Mass Spec Principles

Ionizer

Sample

+_

Mass Analyzer Detector

ESIMALDI

Quadrupole (Q)Iontrap (LT, Orbitrap)TOFFT -ICR

Micro channel platedetector

Importance of Proteins

• they serve as catalysts that maintain metabolic processes in the cell.

• they serve as structural elements both within and outside the cell.

• they are signals secreted by one cell or deposited in the extracellular matrix that are recognized by other cells.

• they are receptors that convey information about the extracellular matrix to the cell.

• they serve as intracellular signaling components that mediate the effects of receptors.

• they are key components of the machinery that determines which genes are expressed and whether mRNAs are translated into proteins.

• they are involved in manipulation of DNA and RNA through processes such as: DNA replication, DNA recombination, RNA splicing or editing.

applications

• What is Omics good for?

• WHAT? Biomarker / Drug target

• Establishing and mining proteome from different species

• At least 200 different PTMs have been identified.

• Expression proteomics• Functional proteomics• Mining: identification of proteins (catalog

the proteome)• Protein-expression profile: identification of

protein interest in a particular state of the tissue/organism

• Protein-network mapping: protein interactions in living systems

• Mapping of protein modifications: how and where proteins are modified.

Sample collection

Proteins extraction

Proteomic analysis

Bioinformatics analysis

Proteomic discovery

Less number of sample size

DiscoveryDetect multiple biomarker candidates

Biomarker discovery

Validation phase Discovery phase

More number of sample size

validation

Functional study

Diagnostics andtherapeutics use

Confirmation of biomarker panels in test patient populations

Identification

The Biomarker Discovery Process

• Proteomic applications in Diabetes

• Helps in protein changes duo to glycation

• molecular mechanisms underpinning disease processes and the effects of treatment

• To understand the impact of: stress, environmental stimuli, food, genetic and obesity, etc.

• Tissue proteomics to understand pathophysiologyand drug target.

• Helps in early diagnosis of diabetic complications.

• identifying new targets for therapeutic development.

• Identification of surrogate markers looking at the plasma/urine/tissue proteome at different stages of development of diabetes, its complications.

• To study the signal transduction network of the insulin receptor and/or other cell surface receptors.

• Characterization of the proteome or a subset of the proteome of animal and cell models

• Identification of novel signaling molecules and pathways involved in cell development, differentiation, communication, function and destruction, etc.

• for studying the regulation, synthesis, secretion, and action of hormones and cytokines

• Proteomic applications in Cancer

• Proteins serve as hallmark for the physiological status of cell

• The ability of physicians to effectively treat cancer is directly dependent on their ability to detect cancers at their earliest stages.

• Proteomic technologies hold recently great promise in the search of new biomarkers for the early detection and the discovery of new therapeutic targets.

• Proteomics technologies to identify unique biosignatures and biomarkers responsible for the diagnosis, prognosis and therapeutic prediction of such disease.

• Biomarkers found in blood, other body fluids, or tissues that are a sign of a normal or abnormal process, or disease. They may also be used to see how well the body responds to a treatment for a disease or condition.

• Other diseases • Pancreatitis• Tress induced poteomics• Eye disease• Cardiac diseases• Smoking • GI diseases• Eclampsias• Prostaties ,• Infections,• Autoimmune diseases, etc….

System biology• Body fluids

• Blood cells

• Erythrocytes

• Leukococytes

• Monocyttes/macrophages

• Lymphocytes

• Platelet

• Plasma and serum

• Urine

• Amniotic fluid

• Cerebrospinal fluid

• Synovial fluid

• Saliva

• Sweat

• Tears

• Semen

• Etc…

• Solid tissues

• Heart

• Brain

• Thyroid

• Muscle

• Malignant

• Tissue culture

• Malignant cells

• Bacterial proteins

Proteins classes for Analysis and characterization

• Membrane

• Soluble proteins

• Nuclear

• Chromosome-associated

• Phosphorylated

• Glycosylated

• Complexes

• genome alone is not sufficient for a complete understanding of complex biological processes.