Tandem peptide identification

The methods for each study are divided into the initial protein separation step, a second separation step if applicable, the type of mass analysis, and the software used for peptide identification. ID = one dimensional polyacrylamide gel electrophoresis, 2D = two dimensional polyacrylamide gel electrophoresis, MS = mass spectrometry (peptide mass fingerprinting), MS/MS = tandem mass spectrometry, MALDI-TOF = matrix assisted laser desorption/ionization-time of flight, MS FIT = software from Protein Prospector, http //prospector.ucsf edu/, ESI = electrospray ionization, Q-TOF = quadrupole-time of flight, PPSS2 =Protana s Proteomic Software Suite (ProtanaEngineering, Odense, Denmark), Mascot = Matiix Science, http //www.matrixscience.com/, TOF-TOF = MALDI plus TOF tandem mass spectrometry, RP-HPLC = reverse phase high performance liquid chromatography, Q-IT = quadrupole ion trap, LIT = linear ion trap. Bioworks = Thermo Electron Corporation. 

L. Li, C D. Masselon, G.A. Anderson, L. Pasa-Tolic, S.-W. Lee, Y. Shen, R. Zhao, M S. Lipton, T.P. Comads, N. Tohc, R.D. Smith, High-throughput peptide identification from protein digests using DDA multiplexed tandem FT-ICR-MS coupled with capillary LC, Anal. Chem., 73 (2001) 3312. 

Ryu, S. Goodlett, D. R. Noble, W. S. Minin, V. N. A Statistical Approach to Peptide Identification from Clustered Tandem Mass Spectrometry Data. Proceedings (IEEE Int. Corf. 

It has been demonstrated also that the iTRAQ tandem mass spectrometric quantitative analysis strategy can be used in conjunction with the quadrupole ion trap by performing multiple stages of mass analysis (that is, MS ) [125], For example, chemical derivatization with the iTRAQ reagent not only labels the N-terminus of a peptide, but the lysine side chain also. Thus, tryptic peptides with a modified lysine residue present at the C-terminus will produce a yj product ion at m/z 291 following ClD-tandem mass spectrometry. To generate the low m/z iTRAQ reporter ions required for quantitation, the yj product ion is isolated and subjected to data-dependent CID-MS. Using this approach, peptide identification is achieved in the MS/MS scan, while quantitation is achieved via MS. ... 

Boyne, M.T., Garcia, B.A., Li, M., Zamdborg, L., Wenger, C.D., Babai, S., Kelleher, N.L. (2009) Tandem mass spectrometry with nltrahigh mass accnracy clarifies peptide identification by database retrieval. J. Proteome Res., 8, 374-379. 

Tabb DL, Fernando CG, Chambers MC. MyriMatch highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis. J Proteome Res. 2007 6 654-61. 

Zhang, Z., Sun, S., Zhu, X., et al. (2006) A novel scoring schema for peptide identification by searching protein sequence databases using tandem mass spectrometry data. BMC Bioinformatics, 7,222. 

Tandem mass spectrometry (MS/MS) is another common approach used for protein identification. In this method, proteins are digested and the resulting peptides are ionized directly from the liquid phase by... 

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 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

Cargile, B. J., Stephenson, J.L., Jr. (2004). An alternative to tandem mass spectrometry isoelectric point and accurate mass for the identification of peptides. Anal. Chem. 76, 267-275. 

Qian, W.J., Liu, T., Monroe, M.E., Strittmatter, E.F., Jacobs, J.M., Kangas, L.J., Petritis, K., Camp, D.G., 2nd, Smith, R.D. (2005b). Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis the human proteome. J. Proteome Res. 4, 53-62. 

The rapid development and sensitivity of the mass spectrometric methods can be foreseen and in the near future the labeling can be more frequently eliminated. The identification of the cross-linked peptide can be detected first with immunological methods and then the digested and cleaved fragments with specific tandem MS techniques. The different photophores hold discrete MS fingerprints, which allow fast recognition of the modified sites. 

Several algorithms are available for the analysis of MS/MS spectra including SEQUEST, MASCOT, and X Tandem among others. Note that additional secondary quality control of assessment of MS/MS data has recently been implemented to assess identification probabilities and false positivity rates. The MS/MS spectra from an experiment can be interrogated against a concatenated forward and reverse database and an assessment of the intrinsic error rate of the data set can be made. Other approaches for secondary analysis of matching scores for peptide sequencing data include XCorr score normalization routines that are independent of peptide and database size.33... 

Tandem MS has emerged as a definitive approach for identifying proteins from multiple sources including complex mixtures. In comparison to PMF, MS/MS permits more definitive identification of proteins. Matching of multiple MS/MS spectra to peptide sequences within the same protein... 

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

Complete sequencing of this peptide requires acquisition of additional tandem mass spectra, preferably MS3 fragmentation, of one of the low-mass y-ions. Because the peptide of interest is derived from a biological source, yet another possibility might be the use of sequence databases, similarly to the previous example. Actually, this approach works very well in this case, allowing identification of the peptide of interest as H-LGEYGFQNALIVR-OH, the 421-433 fragment of bovine serum albumin. 

Even though the MALDI peptide mass mapping technique is very powerful, it has limitations. It requires well-separated proteins, is less sensitive than identifications based on electrospray tandem mass spectrometry, can only identify proteins whose complete sequences are available in databases, and does not produce redundant information. 

Fig. 7. Protein identification with electrospray tandem mass spectrometry and a triple quadrupole mass spectrometer. Fragment spectra of several peptides are generated during one investigation. From the fragment spectra short sequence stretches can be read. Together with their mass location in the peptide of the measured mass, they can be used to specifically identify a protein in the database. Because the protein identification depends only on one peptide, several proteins can be identified from one sample.

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