Protein sequence analysis tandem mass spectrometry

Bonner, R., Emmert-Buck, M., Cole, K., Pohida, T., Chuaqui, R., Goldstein, S. and Liotta, L., Laser capture miciodissection molecular analysis of tissue. Science, 278, 1481-1483 (1997). Hunt, D.F., Yates, J.R., 3rd, Shabanowitz, J., Winston, S. and Hauer, C.R., Protein sequencing by tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A., 83, 6233-6237 (1986). 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

Coon JJ, Syka JEP, Shabanowitz J, Hunt DF. Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques 38, 519-523, 2005. 

Although reliable, this technique may lead to false positive results in some cases. To overcome this problem many proteomic companies are now adopting the technique of tandem mass spectrometry to unambiguously identify protein sequences. This technique subjects proteins to successive routines of fragmentation and mass analysis in order to provide the actual amino acid sequence. 

Tandem mass spectrometry coupled to electrospray ion source allowed not only to identify proteins but also to characterize post-translational modifications of a protein (phosphorylation, acetylation, methylation, glycosylation, etc.). Indeed, the presence of such modifications induces an increase of the peptide molecular masses compared to the calculated masses based on the theoretical sequence, which often directly identifies the type of modification. In addition, tandem mass spectrometry allows in general precise localization of the modification at specific residue of the peptide. Analysis of modifications allows to understand biological mechanisms because several processes are controlled and/or induced by such modifications (Mann and Jensen 2003). Being... 

The basic goal of the mass spectrometry measurement in the context of peptide analysis in proteomics and phosphoproteomics is to determine specific attributes that are then used in subsequent database searches to provide 1. the identity of the proteins present in the sample 2. location of the site(s) of phosphorylation in these proteins. Both pieces of information are derived from the mass of the peptide and, most importantly, from the gas-phase dissociation patterns that are diagnostic of the peptide s amino acid sequence and phosphosite location. The gas-phase dissociation patterns are obtained via tandem mass spectrometry (MS/MS). On a phosphoproteome-wide scale, the analysis includes measurement of the attributes for many thousands of individual peptides. 

Edman degradation was originally developed for determination of the primary structure (i.e., amino acid sequence) of peptides and proteins. Sequence analysis is not regularly performed for quality control in routine peptide synthesis but is more often employed for problem solving. As described earlier in this chapter, efficient characterization of synthetic peptides can be readily obtained by a combination of RP-EDPLC and mass spectrometry. Amino acid analysis is also valuable if MS is not available. If an incorrect mass or a discrepancy in the amino acid composition is found, one obvious alternative is to resynthesize the peptide. But, in order to deduce the cause of a failed synthesis, additional analyses must be performed. Both Edman degradation and tandem MS can be used to obtain sequence information... 

PRIMARY STRUCTURE OF PROTEINS SEQUENCE ANALYSIS BY TANDEM MASS SPECTROMETRY 101... 

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