Protein identification, by mass

Griffin, T. J. Han, D. K. Gygi, S. R Rist, B. Lee, H. Aebersold, R. Parker, K. C. Toward a high-throughput approach to quantitative proteomic analysis Expression-dependent protein identification by mass spectrometry. J. Am. Soc. Mass. Spectrom. 2001,12,1238-1246. 

Protein identification by mass spectrometry consists of three stages ionization, separation and detection. However, this will not be discussed in depth here for an explanation of the technical aspects of modern mass spectrometry please refer to Yates (1998) or Mann et al. (2001) amongst others. 

Liska, A.J. and Shevchenko, A. (2003) Expanding the organismal scope of proteomics cross-species protein identification by mass spectrometry and its implications. Proteomics 3, 19-28. 

Figure 15.6 Schematic depiction of one example of protein identification by mass spectrometry. Genes of interest are tagged, then transfected into mammalian cells, and proteins associated with the cognate tagged protein are purified by affinity methods. Separation of the complex is carried out by 2D SDS-PAGE. Identification of the proteins is by MALDI and ESI (Blackwell, 1999).

James P, Quadroni M, Carafoli E, Gonnet G. Protein identification by mass profile fingerprinting. Biochem Biophys Res Comm 1993 195(l) 58-64. 

Protein Identification by Mass Spectrometric Analyses of Peptides... 

Fluorescent dye comes in a variety of choices they are quick and easy to use and are highly sensitive like Silver stain but not compatible to subsequent techniques of protein identification by mass spectroscopy. 

In this chapter we will review proteomic investigations of cardiac proteins and focus on their application to the study of heart disease in the human and in animal models of cardiac dysfunction. The majority of these studies of the cardiac proteome have involved protein separation, visualisation and quantitation using the traditional 2-DE approach combined with protein identification by mass spectrometry. These essential technologies will be briefly described. However, there is increasing interest in using alternative gel-free techniques based on mass spectrometry or protein arrays for high throughput proteomics. These alternative approaches will be introduced, but further details can be found in Chapter 2 of this volume by Michel Faupel. 

Rabilloud, T., Kieffer, S., Procaccio, V., Louwagie, M., Courchesne, P.L., Patterson, S.D., Martinez, P., Garin, J., and Lunardi, J., 1998, Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry toward a human mitochondrial proteome. Electrophoresis. 19 1006-1014. 

Fig. 1. Schematic representation of protein identification by mass spectrometry and preparation of reference 2D maps.

Figure 3 Protein identification by mass spectrometry, in a typicai strategy, digested peptides are anaiyzed by MALDi-TOF-MS in order to determine the masses of intact peptides. These masses can be used in correiative database searches to identify exact matches. If this approach fails, ESI-MS/MS analysis can be used to generate peptide fragment ions. These can be used to search less robust data sources and to produce de novo peptide sequences. (Reproduced with permission from Twyman RM (2004) Principles of Proteomics. Abington, UK Bios/Garland Publishers.)...

For a scale of purification that would be enough for protein identification by mass spectrometry, about 30 g fresh tissue or 50 g suspension-cultured cells (fresh weight) should be used. For suspension-cultured cells, grinding with acid washed sand may be necessary. 

Lubec, G. Afiew-Sadat, L. Limitations and pitfalls in protein identification by mass spectrometry. Chem. Rev. 2007, 107, 3568-3584. 

The Classical Approach 2-DE Separation and Protein Identification by Mass Spectrometry... 

---