Proteome and proteomics

Introduction
Proteome is the entire set of proteins that is expressed by genome, cell, tissues or organisms.
Among other tasks, proteins give organisms their structure, they transport molecules, and they take care
of cell signaling. Proteins are even responsible for creating proteins when and where they are needed
and disassembling them when they are no longer required. Monitoring proteins is therefore essential to
understanding any biological system, and proteomics is the discipline tasked with achieving this.

Proteomics is branch of molecular biology concerned with proteome and the term proteomic
was coined in 1994 by marc wilkins. Proteomic simply defined as the study of protein particularly their
structures, expression and functions or we can say that proteomic is the systematic study of all of the
proteins in a cell, tissues or organisms.

Branches of proteomics
Functional proteomics
Functional proteomics aims for the elucidation of the biological function of proteins or protein
groups and classes on a proteome-wide level. This includes the characterization of enzyme activities as
well as protein/protein interactions and post-translational modifications at proteins. Combining our
technologies and expertise, we are able to map changes in signaling pathways as a consequence of cell-
treatment or changes in environmental conditions. This can be done either by the large-scale
measurement of enzymatic activities of kinase, phosphatase, protease or other protein modifying
enzymes or by measurement of epigenetic modifications at proteins.

Quantitative proteomics
Quantitative proteomics is an analytical chemistry technique for determining the amount
of proteins in a sample the methods for protein identification are identical to those used in general (i.e.
Qualitative) proteomics, but include quantification as an additional dimension. Rather than just
providing lists of proteins identified in a certain sample, quantitative proteomics yields information
about the physiological differences between two biological samples. For example, this approach can be
used to compare samples from healthy and diseased patients. Quantitative proteomics is mainly
performed by two-dimensional gel electrophoresis (2-de) or mass spectrometry (ms). However, a recent
developed method of quantitative dot blot (qdb) analysis is able to measure both the absolute and
relative quantity of an individual proteins in the sample in high throughput format, thus open a new
direction for proteomic research. In contrast to 2-de, which requires ms for the downstream protein
identification, ms technology can identify and quantify the changes.
Expression proteomics
Expression proteomics includes the analysis of protein expression at larger scale. It helps
identify main proteins in a particular sample, and those proteins differentially expressed in related
samples—such as diseased vs. Healthy tissue. If a protein is found only in a diseased sample then it can
be a useful drug target or diagnostic marker. Proteins with same or similar expression profiles may also
be functionally related. There are technologies such as 2d-page and mass spectrometry that are used in
expression proteomics.

Proteomic workflow
 1st sample preparations
 Extract protein from cell
 Use 2d-electrophoresis method to separate different proteins
 Than try to cut protein into peptides
 Use mass spectrometry for peptide detections
 Than interpretation all the data

Protein isolation methods

There are three main methods for protein isolation from cell, tissue, and organ. The initial steps of
protein extraction often involve crude mechanical disruption such as cutting, smashing, or shearing
tissue into smaller pieces. If intracellular proteins are the target, then detergents can be used to help
break apart the phospholipid cellular membrane (cell lysis). Sonication is also used to disrupt the
membrane. Often, protease inhibitors are added to the mix to prevent loss of protein due to enzymatic
degradation.

A.Cryogenic grinding ( grinding in liquid nitrogen )

1. Used for hard tissues and cells like roots, stems but also for hard walled cell like algae and
cynobacteria
2. Low temperature protect the protein during grinding
3. Time consuming method
4. Require suitable machinery

B. Ultrasound homogenization
1. Used for soft tissues like some leaves and post treatment after grinding
2. Does not requiring freezing
3. Used for individual cells (plant cell culture, algae or bacteria)
 4. Requires very expensive machinery

C. lysis buffer
1. Used only for bacteria and animals cell
2. May cause degradation
3. No machinery required
4. Most commonly used method
5. Used different buffer solution such as NACL, ACK, Tris-Hcl, SDS, EDTA

Traditional routes of protein study

In proteomics, there are multiple methods to study proteins. Generally, proteins may be detected
by using either antibodies (immunoassays) or mass spectrometry. If a complex biological sample is
analyzed, either a very specific antibody needs to be used in quantitative dot blot analysis (qdb), or
biochemical separation then needs to be used before the detection step, as there are too many analytes
in the sample to perform accurate detection and quantification.

Protein detection with antibodies (immunoassays)

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 molecular biologists today.
There are several specific techniques and protocols that use antibodies for protein detection. The
enzyme-linked immunosorbent assay (elisa) has been used for decades to detect and quantitatively
measure proteins in samples. The western blot may be used for detection and quantification of individual
proteins, where in an initial step, a complex protein mixture is separated using sds-page and then the
protein of interest is identified using an antibody.

Modified proteins may be studied by developing an antibody specific to that modification. For
example, there are antibodies that only recognize certain proteins when they are tyrosine-
phosphorylated, they are known as phospho-specific antibodies. Also, there are antibodies specific to
other modifications. These may be used to determine the set of proteins that have undergone the
modification of interest.

Antibody-free protein detection

While protein detection with antibodies is still very common in molecular biology, other
methods have been developed as well, that do not rely on an antibody. These methods offer various
advantages, for instance they often are able to determine the sequence of a protein or peptide, they may
have higher throughput than antibody-based, and they sometimes can identify and quantify proteins for
which no antibody exists. Some of the methods are given bellow.
Detection methods
One of the earliest methods for protein analysis has been edman degradation (introduced in
1967) where a single peptide is subjected to multiple steps of chemical degradation to resolve its
sequence. These early methods have mostly been replaced by technologies that offer higher throughput.

More recently implemented methods use mass spectrometry-based techniques, a development

that was made possible by the discovery of "soft ionization" methods developed in the 1980s, such as
matrix-assisted laser desorption/ionization (maldi) and electrospray ionization (esi). These methods
gave rise to the top-down and the bottom-up proteomics workflows where often additional separation
is performed before analysis.

Mass spectrometry methods

 There are two mass spectrometry based methods currently used for protein profiling.

 The more widespread method uses high resolution 2d electrophoresis to separate protein from
different samples.
 It followed by staining of differentially expressed protein to be identify by spectrometry.
Application of proteomics

Discovery of protein biomarkers

Biomarkers of drug efficacy and toxicity are becoming a key need in the drug development
process. Mass spectral-based proteomic technologies are ideally suited for the discovery of protein
biomarkers in the absence of any prior knowledge of quantitative changes in protein levels. The success
of any biomarker discovery effort will depend upon the quality of samples analysed, the ability to
generate quantitative information on relative protein levels and the ability to readily interpret the data
generated. This review will focus on the strengths and weaknesses of technologies currently utilized to
address these issues

Study of tumor metastasis.

Tumor metastasis is the dominant cause of death in cancer patients. However, the molecular
and cellular mechanisms underlying tumor metastasis are still elusive. The identification of protein
molecules with their expressions correlated to the metastatic process would help to understand the
metastatic mechanisms and thus facilitate the development of strategies for the therapeutic interventions
and clinical management of cancer. Proteomics is a systematic research approach aiming to provide the
global characterization of protein expression and function under given conditions. Proteomic
technology has been widely used in biomarker discovery and pathogenetic studies including tumor
metastasis. combination of proteomics with other experimental approaches in biochemistry, cell
biology, molecular genetics and chemistry, together with the development of new technologies and
improvements in existing methodologies will continue to extend its application in studying cancer
metastasis.

Fetal and maternal medicine

The recent elucidation of the human genome sequence has provided a wealth of useful
information but does not provide information on diseases caused by changes at the protein level.
Proteomics includes the characterization and functional analysis of all proteins that are expressed by the
genome at a certain moment, under certain conditions. Since expression levels of many proteins strongly
depend on complex, but well-balanced regulatory systems, the proteome, unlike the genome, is highly
dynamic. This variation depends on the biological function of a cell, but also on signals from its
environment. In (bio) medical research it has become increasingly apparent that cellular processes, in
particular in disease, are determined by multiple proteins. Hence it is important not to focus on one
single gene product (one protein), but to study the complete set of gene products (the proteome). In this
way the multi-factorial relations underlying certain diseases may be unraveled potentially identifying
therapeutically targets. For many diseases characterization of the functional proteome is crucial for
elucidating alterations in protein expression and modifications. When proteins undergo non-genetically
determined alterations such as alternative splicing, or post-translational modifications, e.g.
Phosphorylation or glycosylation, it may affect their function. Although abnormalities in splicing or
post-translational modifications can cause a disease process, they can also be a consequence. An
example is that patients with diabetes have high blood glucose which glycosylates hundreds or even
thousands of proteins, including hba1c which is used to monitor diabetic control.

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