Proteomics1

Proteomics is the large-scale study of proteins.

Proteins are vital parts of living organisms, with many functions.

In proteomics, there are multiple methods to study proteins. Generally, proteins can either be detected using antibodies (immunoassays) or using mass spectrometry. If a complex biological sample is analyzed, then biochemical separation has to be used before the detection step as there are too many analytes in the sample to perform accurate detection and quantification.

 

Protein detection using 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 can 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 can 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, known as phospho-specific antibodies. 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.

 

Antibody-free protein detections

 

While protein detection with antibodies are still very common in molecular biology, also other methods have been developed that do not rely on an antibody. These methods offer various advantages, for instance they are often 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.

 

Detection method

 

One of the earliest method for protein analysis where a single peptide is subjected to multiple steps of chemical degradation to resolve its sequence. These methods have mostly been supplanted by technologies that offer higher throughput.

More recent methods use mass spectrometry-based techniques, a development that was made possible by the discovery of "soft ionization" methods such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) developed in the 1980s. These methods gave rise to the top-down and the bottom-up proteomics workflows where often additional separation is performed before analysis (see below).

 

Separation method

 

For the analysis of complex biological samples, a reduction of sample complexity is required. This can be performed off-line by one-dimensional or two dimensional separation. More recently, on-line methods have been developed where individual peptides (in bottom-up proteomics approaches) are separated using Reversed-phase chromatography and then directly ionized using ESI; the direct coupling of separation and analysis explains the term "on-line" analysis.

 

Hybrid technology

 

There are several hybrid technologies that use antibody-based purification of individual analytes and then perform mass spectrometric analysis for identification and quantification. Examples of these methods are the MSIA (mass spectrometric immunoassay)  and the SISCAPA (Stable Isotope Standard Capture with Anti-Peptide Antibodies) method.

 

 

biomarker

 

-> characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.

Understanding the proteome, the structure and function of each protein and the complexities of protein–protein interactions is critical for developing the most effective diagnostic techniques and disease treatments in the future. For example, proteomics is highly useful in identification of candidate biomarkers (proteins in body fluids that are of value for diagnosis), identification of the bacterial antigens that are targeted by the immune response, and identification of possible immunohistochemistry markers of infectious or neoplastic diseases.

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. Secretomics, a subfield of proteomics that studies secreted proteins and secretion pathways using proteomic approaches, has recently emerged as an important tool for the discovery of biomarkers of disease.

 

 

unknown Protein identification

 

Mass spectrometry and microarray produce peptide fragmentation information but do not give identification of specific proteins present in the original sample. Due to the lack of specific protein identification, past researchers were forced to decipher the peptide fragments themselves. However, there are currently programs available for protein identification. These programs take the peptide sequences output from mass spectrometry and microarray and return information about matching or similar proteins. This is done through algorithms implemented by the program which perform alignments with proteins from known databases such as UniProt  and PROSITE  to predict what proteins are in the sample with a degree of certainty.

 

Reference

https://en.wikipedia.org/wiki/Proteomics

 

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