Introduction

Proteomics is a key tool to study proteins, particularly their structures and functions and from there to understanding of healthy and diseased conditions. It involves the identification of all the proteins made in a given cell, tissue or organism, understanding how these proteins function and the determination of their 3D structures. This information can be used to help identify target sites for drug interaction. 

Definition of Proteomics

The study of the proteome, the complete set of proteins produced by a species, using the technologies of large-scale protein separation and identification. The term proteomics was coined in 194 by Marc Wilkins who defined it as "the study of proteins, how they're modified, when and where they're expressed, how they're involved in metabolic pathways and how they interact with one another."

Proteomic Techniques

A typical cell produces hundreds of thousands of different proteins. Proteomics is much more complicated than genomics because while an organism's genome is more or less constant, the proteome differs from cell to cell and from time to time. This is because distinct genes are expressed in distinct cell types. This means that even the basic set of proteins which are produced in a cell needs to be determined. In the past this was done by mRNA analysis, but this was found not to correlate with protein content. It is now known that mRNA is not always translated into protein, and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.

Understanding the proteom gives a much better understanding of an organism than genomics.
1. The level of transcription of a gene gives only a rough estimate of its level of expression into a protein. An mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a small amount of protein.
2. Many proteins experience post-translational modifications that profoundly affect their activities; for example some proteins are not active until they become phosphorylated. Methods such as phosphoproteomics and glycoproteomics are used to study post-translational modifications.
3. Many transcripts give rise to more than one protein, through alternative splicing or alternative post-translational modifications.
4. Many proteins form complexes with other proteins or RNA molecules, and only function in the presence of these other molecules.
5. Protein degradation rate plays an important role in protein content.

 Methods to study Proteins

1. Determining proteins which are post-translationally modified

One way in which a particular protein can be studied is to develop an antibody which is specific to that modification. For example, there are antibodies which only recognize certain proteins when they are tyrosine-phosphorylated; also, there are antibodies specific to other modifications. These can be used to determine the set of proteins that have undergone the modification of interest.

For sugar modifications, such as glycosylation of proteins, certain lectins have been discovered which bind sugars. These too can be used.

A more common way to determine post-translational modification of interest is to subject a complex mixture of proteins to electrophoresis in "two-dimensions", which simply means that the proteins are electrophoresed first in one direction, and then in another... this allows small differences in a protein to be visualized by separating a modified protein from its unmodified form. This methodology is known as "two-dimensional gel electrophoresis".

Recently, another approach has been developed called PROTOMAP which combines SDS-PAGE with shotgun proteomics to enable detection of changes in gel-migration such as those caused by proteolysis or post translational modification.

2. Determining the existence of proteins in complex mixtures

Classically, antibodies to particular proteins or to their modified forms have been used in biochemistry and cell biology studies. These are among the most common tools used by practicing biologists today.

For more quantitative determinations of protein amounts, techniques such as ELISAs can be used.

For proteomic study, more recent techniques such as Matrix-assisted laser desorption/ionization have been employed for rapid determination of proteins in particular mixtures.


3. Establishing protein-protein interactions

Most proteins function in collaboration with other proteins, and one goal of proteomics is to identify which proteins interact. This is especially useful in determining potential partners in cell signaling cascades.

Several methods are available to probe protein-protein interactions. The traditional method is yeast two-hybrid analysis. New methods include protein microarrays, immunoaffinity chromatography followed by mass spectrometry, dual polarisation interferometry and experimental methods such as phage display and computational methods.

Practical applications of proteomics

One of the most promising developments to come from the study of human genes and proteins has been the identification of potential new drugs for the treatment of disease. This relies on genome and proteome information to identify proteins associated with a disease, which computer software can then use as targets for new drugs. For example, if a certain protein is implicated in a disease, its 3D structure provides the information to design drugs to interfere with the action of the protein. A molecule that fits the active site of an enzyme, but cannot be released by the enzyme, will inactivate the enzyme. This is the basis of new drug-discovery tools, which aim to find new drugs to inactivate proteins involved in disease. As genetic differences among individuals are found, researchers expect to use these techniques to develop personalized drugs that are more effective for the individual.

A computer technique which attempts to fit millions of small molecules to the three-dimensional structure of a protein is called "virtual ligand screening". The computer rates the quality of the fit to various sites in the protein, with the goal of either enhancing or disabling the function of the protein, depending on its function in the cell. A good example of this is the identification of new drugs to target and inactivate the HIV-1 protease. The HIV-1 protease is an enzyme that cleaves a very large HIV protein into smaller, functional proteins. The virus cannot survive without this enzyme; therefore, it is one of the most effective protein targets for killing HIV.

Biomarkers

Understanding the proteome, the structure and function of each protein and the complexities of protein-protein interactions will be critical for developing the most effective diagnostic techniques and disease treatments in the future.

An interesting use of proteomics is using specific protein biomarkers to diagnose disease. A number of techniques allow to test for proteins produced during a particular disease, which helps to diagnose the disease quickly. Techniques include western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA) or mass spectrometry. The following are some of the diseases that have characteristic biomarkers that physicians can use for diagnosis.

Alzheimer's disease

In Alzheimer’s disease, elevations in beta secretase create amyloid/beta-protein, which causes plaque to build up in the patient's brain, which is thought to play a role in dementia. Targeting this enzyme decreases the amyloid/beta-protein and so slows the progression of the disease. A procedure to test for the increase in amyloid/beta-protein is immunohistochemical staining, in which antibodies bind to specific antigens or biological tissue of amyloid/beta-protein.

Heart disease

Heart disease is commonly assessed using several key protein based biomarkers. Standard protein biomarkers for CVD include interleukin-6, interleukin-8, serum amyloid A protein, fibrinogen, and troponins. cTnI cardiac troponin I increases in concentration within 3 to 12 hours of initial cardiac injury and can be found elevated days after an acute myocardial infarction. A number of commercial antibody based assays as well as other methods are used in hospitals as primary tests for acute MI.

Separation techniques used in Proteomics

Enzymatic digestion (eg with trypsin) of extracted proteins generates a range of peptides in the digest that can be separated by 2D gel electrophoresis, .

 Multidimensional LC-MS/MS can be used to separate and sequence these peptides.

The 2D HPLC approach increases the resolving power of the separation, by combining reversed-phase with ion-exchange, HILIC or affinity techniques. This reduces the complexity of the peptide mixture being analysed, improving detection chances for less abundant species.

Due to the limited amounts of sample generally available, microbore and capillary/nano columns are now preferred. Experimental electrospray MS/MS data is matched against sequence-analysis identification software.

Many products listed on this web-site can be used for proteomic studies.

HPLC Columns

Capillary and LC-MS columns are discussed further . In addition to reversed-phase columns, ion-exchange, HILIC  and affinity columns are also used for 2D analyses.
A selection of suitable columns can be found on the following pages.

Reversed-phase columns: ACE , Inertsil , Kromasil, NUCLEOSIL , Vydac, YMC, Zorbax

Ion-exchange columns: BioBasic , PolyLC, Shodex, TSKgel

HILIC columns: Inertsil HILIC, PolyLC, TSKgel Amide-80, ZIC-HILIC

Affinity columns: Multiple Removal System, Shodex, TSKgel , Venture A

Speciality columns: Titansphere  - especially effective in the isolation of phosphopeptides.

Tryptic Digest Standards and Kits

A range of protein tryptic digest test mixtures is available as calibration standards for LC-MS and MALDI-TOF . A 2D Proteomics HPLC kit includes a C18 column, a ZIC-HILIC column and tryptic digest standards.

Sample Preparation

In addition to the conventional SPE products, more specialized products have applications for sample pre-treatment of low level proteins and peptides.

Pipette and syringe tips can separate very low sample volumes, prior to HPLC, MALDI and electrophoresis. The following material tips are available: PolyLC, ZIC-HILIC,  Titania, Monolithic.

Switching Valves

The Rheodyne MX Series II automated switching valves can be used in proteomic applications