Peptides sequencing, by tandem mass spectrometry

J. A. Taylor and R. S. Johnson. Sequence Database Searches via de novo Peptide Sequencing by Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom., 11, no. 9 (1997) 1067-1075. 

Johnson R.S. and Taylor J.A. (2000), Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry, in Methods in Molecular Biology, Vol. 146, Mass Spectrometry of Proteins and Peptides, pp. 41-61, Chapman J.R., Ed., Humana Press, Totowa, NJ. 

Taylor, J. A., and Johnson, R. S. (2001). Implementation and uses of automated de novo peptide sequencing by tandem mass spectrometry. Anal. Chem. 73 2594-2604. 

Sequence ion analysis of each cleaved peptide fragment by tandem mass spectrometry (MS/MS). 

Ma, B., Zhang, K., Hendrie, C., Liang, C., Li, M., Doherty-Kirby, A. and Lajoie, G., PEAKS powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom., 17, 2337-2342 (2003). 

Although the most practiced method for MS-based protein sequencing combines proteolytic cleavage of the intact protein with subsequent sequencing of the resulting peptide fragments by tandem mass spectrometry, especially with LC-MS/MS and MAL-DI-MS/MS, there is value in the direct measurement and fragmentation of intact proteins. 

Like peptide oligomers, peptoids can be analyzed by HPLC and by mass spectrometry. They can be sequenced by Fdman degradation [13] or by tandem mass spectrometry [14] since, like polypeptides, they conveniently fragment along the main chain amides [15, 16]. 

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. 

Sequence Analysis of Antimicrobial Peptides by Tandem Mass Spectrometry... 

M. F. Bean and S. A. Carr, Characterization of disnlfide bond position in proteins and sequence analysis of cysteine-containing peptides by tandem mass spectrometry. Anal. Biochem. 201, 216-226 (1992). 

Biemann, K. (1990) Sequencing of peptides by tandem mass spectrometry and high-energy collision-induced dissociation. Methods Enzymol., 193, 455-479. 

Figure 13 Comparative peptide mapping of two forms of rSmp28 by LC-MS (postcolumn stream splitting see Fig. 3). (A) Analysis of the nonmodified and (B) of the modified protein ( labels the nonmodified and O the modified N-terminal peptide). The on-line mass spectra of the nonmodified (C) and the modified (D) N-terminal peptides indicate a difference of 26.1 Da, and amino acid composition analysis of the collected peptide fractions gave identical results (expected sequence AGEHIK). The modified peptide was inaccessible to Edman degradation, lacked two deuterium-exchangeable hydrogen atoms, and could be separated into two isomers. Detailed analysis by tandem mass spectrometry showed that it was modified due to addition of acetaldehyde to a H, and K (see Fig. 14 and Ref. 44). (From Ref. 44.)...

Nowadays, sequencing of peptides and other biopolymers by tandem mass spectrometry represents a major field of work for many mass spectrometrists [136-139] FAB and LSIMS no longer play a role here (for an example of peptide sequencing by FAB-MS cf. Chap. 9.6.6). 

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. 

Tandem mass spectrometry (MS/MS) is a method for obtaining sequence and structural information by measurement of the mass-to-charge ratios of ionized molecules before and after dissociation reactions within a mass spectrometer which consists essentially of two mass spectrometers in tandem. In the first step, precursor ions are selected for further fragmentation by energy impact and interaction with a collision gas. The generated product ions can be analyzed by a second scan step. MS/MS measurements of peptides can be performed using electrospray or matrix-assisted laser desorption/ionization in combination with triple quadruple, ion trap, quadrupole-TOF (time-of-flight), TOF-TOF or ion cyclotron resonance MS. Tandem... 

The major advantage of the tandem mass spectrometry approach compared to MALDI peptide fingerprinting, is that the sequence information obtained from the peptides is more specific for the identification of a protein than simply determining the mass of the peptides. This permits a search of expressed sequence tag nucleotide databases to discover new human genes based upon identification of the protein. This is a useful approach because, by definition, the genes identified actually express a protein. 

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

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