Protein Characterisation in Proteomics

Abstract

Proteomics refers to the comprehensive analysis of proteomes – the protein content of complex biological mixtures, that is, a whole proteome of a cell, microbe or a proteome of a certain tissue or just the proteome of the blood, saliva or cerebrospinal fluid. The aim is to separate and identify all the proteins of the chosen proteome and then to determine their interactome and with that their biological relevance and activity in that system at the moment of sample collection. Currently, such analyses would also include the characterisation of possible secondary modifications and determining the expression level of each protein. All this is performed by high‐end mass spectrometers that allow the identification and parallel quantification of several hundred proteins and several of their secondary modifications (e.g. phosphorylations, sumoylations, glycosylations etc.) in one analysis step. Thus, a big challenge is also to be able to deal with an enormous amount of complex data in order to make any sense of the results at the end. In this article, the authors will highlight some key issues for such analyses and describe some of the used technologies in greater detail.

Key Concepts:

  • Proteins are one of the key elements of life.

  • Proteome is the full content of proteins in a given biological sample.

  • Proteome content can change dynamically according to, for example, time, location and environmental conditions.

  • Meaningful results from proteomic analysis can be obtained if the analysis is restricted to a well‐defined and targeted goal.

  • Main methods include two‐dimensional gel separation, enzyme digestion and multidimensional liquid chromatography mass spectrometry.

  • Along the flow of proteomic analysis it is important to choose the most appropriate approach (top‐down vs. bottom‐up, focused vs. broad, quantitative vs. qualitative, comparative, selective).

  • Protein analysis can be subjected to positive and negative selection procedures for desired features.

  • Post‐translational modifications can change protein characteristics and function.

  • Large depositories of data are available for comparative analysis.

Keywords: proteome; post‐translational modifications; mass spectrometry; matrix assisted laser desorption ionisation (MALDI); electrospray ionisation (ESI); isobaric tag labelling for relative and absolute quantitation (ITRAQ); isotope coded affinity tag labelling (ICAT); stable isotope labelling by amino acids (SILAC); label‐free quantitation; surface plasmon resonance (SPR)

Figure 1.

An example of depleted sample (a) and a nondepleted sample (b).

Figure 2.

Pre‐fractionation of secondary modifications (PTM's) by selective affinities by, for example, glycoproteins, phosphoproteins, peptides, domains, ligands, antibodies, charges, hydrophobicity, metal affinity etc.

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Web Links

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How to Cite close
Meri, Seppo, and Baumann, Marc(Oct 2012) Protein Characterisation in Proteomics. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0006217.pub2]