Protein/peptide analysis

Multidimensional separations for protein/peptide analysis in the post-genomic era. Biotechniques 2002, 32, 898-911. 

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

Table 5.5 Nomenclature of the ions formed in the mass spectral fragmentation of polypeptides. From Chapman, J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. Reproduced by permission of Humana Press, Inc. 

J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. 

PROTEINS PEPTIDES MUST BE PURIFIED PRIOR TO ANALYSIS... 

DEAE 25W were operated in tandem to map the peptides generated on tryptic hydrolysis of very large proteins, such as albumin genetic variants.197 A useful review that covers many aspects of peptide analysis is available.198... 

Peptide and Protein Drug Analysis, edited by Ronald E. Reid... 

Murphy, R.E. (2001). Comprehensive two-dimensional liquid chromatography for comparative protein analysis. Presented at the International Symposium on the Separation of Proteins, Peptides and Polynucleotides. (ISPPP 2001) Orlando, FL. 

Figure 12.5 illustrates a typical problem of analysis of minor components present in a matrix of highly abundant ones. Despite the availability of large LC-MS peak capacity (Table 12.3), the number of peptides detected in a semm/plasma digest does not exceed several hundreds (Kapp et al., 2005). These peptides typically match 30-50 high abundant proteins. We believe that the maj ority of remaining proteins/peptides in the sample are present at concentrations well below the LOD of MS instrument. 

Janini, G.M., Chan, K.C., Conrads, T.P., Issaq, H.J., Veenstra, T.D. (2004). Two-dimensional liquid chromatography-capillary zone electrophoresis—sheathless electrospray ionization-mass spectrometry evaluation for peptide analysis and protein identification. Electrophoresis 25, 1973-1980. 

As well as protein expression analysis, proteomics in its broadest sense also includes protein-protein interaction mapping. This can range from small studies using a single protein or peptide to fish ouf any proteins that interact with it... 

Kalkhof, S. et al. (2005a) Chemical cross-linking and high-performance fourier transform ion cyclotron resonance mass spectrometry for protein interaction analysis Application to a calmodulin/target peptide complex. Anal. Chem. 77, 495-503. 

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