Proteins peptide sequencing using mass

Fairwell, T., S. Ellis, and R.E. Lovins, Quantitative protein sequencing using mass spectrometry thermally induced formation of thiohydantoin amino acid derivatives from N-methyl- and N-phenylthiourea amino acids and peptides in the mass spectrometer. Anal Biochem, 1973. 53(1) 115-23. 

The development and use of various protein sequence databases for automated search routines (Eng et al., 1994 Clauser et al., 1999) are an essential component of protein analysis that uses mass spectrometry techniques. These programs (i.e., SEQUEST, MASCOT) require only a few peptides for matching therefore, the absence of a match for a particular peptide does not affect the search performance. Using protein database searches provides an efficient way of confirming a putative sequence from corresponding full-scan mass spectrometry and MS/MS data. 

Figure 15.14. Determination of peptide sequence using nanoelectrospray ionization, and a very high-resolution mass analyzer (Q-TOF). In the first quadrupole, a doubly charged peptide ion of m/z — 625.41 was selected and later fragmented. The m/z CID spectrum yields the FGDYGSIDYGR sequence, shown at the top.23 [Reprinted, with permission, from E. Gustafsson, K. Thoren, T. Larsson, P. Davidsson, K. Karlsson, and C. L. Nilsson, Identification of Proteins from Escherichia coli Using Two-Dimensional Semi-Preparative Electrophoresis and Mass Spectrometry. Rapid Communications in Mass Spectrometry 15, 2001, 428-432. Copyright 2001 John Wiley Sons, Ltd.]...

The most common proteomics approach uses two-dimensional gel electrophoresis to separate cellular proteins, followed by in-gel tryptic digestion of the protein spots and identification of the peptide sequences by mass spectrometry. There are s ificant problems with this approach, which so far, have limited its usefulness for drug taiget discovery. A number of protein classes such as int ral membrane proteins, positively chaiged and hydrophobic proteins are difficult to separate. There are often selective losses of individual protdns, for reasons that are not well understood, so the claim for a global picture is not entirefy accurate. The approach is semi-quantitative at best. Importantly, low abundance proteins may not be detected. However, newer approaches are constantly being developei Approaches which... 

In another application, UHPLC-MS technology was developed for rapid comparison of a candidate biosimilar to an innovator monoclonal antibody (mAb) (37). In this study, UHPLC-MS was developed for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for mAb products. Although the biosimilar product is expected to have the same amino acid sequence and modifications as the innovator s product, the observed intact mass by UHPLC-MS was different for the biosimilar compared to the innovator protein. Peptide mapping using UHPLC-MS/MS (38) revealed that the mass difference between the biosimilar and the innovator s product was due to a two amino acid residue variance in the heavy chain sequence of the biosimilar (Figure 8.6). 

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Tandem mass spectrometry (MS/MS) produces precise structural or sequence information by selective and specific induced fragmentation on samples up to several thousand Daltons. For samples of greater molecular mass than this, an enzyme digest will usually produce several peptides of molecular mass suitable for sequencing by mass spectrometry. The smaller sequences can be used to deduce the sequence of the whole protein. 

Mascot (http //www.matrixscience.com/), a search engine that uses mass spectrometry data to identify peptides and proteins from primary sequence databases (MSDB, SwissProt, and others). 

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