Peptide and Protein Identification

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. 

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. 

Chaurand P, Luetzenkirchen F, Spengler B (1999) Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry. J Am Soc Mass Spectroml0 91-103... 

Gradually, over the past twenty years, mass spectrometers were interfaced with a number of protein chemistry assays to generate detectors providing superior information. With the increased performance and versatility of the instrumentation dedicated to the life sciences, new analytical strategies for peptide and protein identification and characterization have emerged in which MS and bioinformatic tools are key players. MS has an enormous impact on the capability for structural analysis of bio-molecules, thanks to the ability to create gas phase ions of the peptides and proteins to be analyzed. Peptides and proteins are often charged and polar, making... 

Macht, M., Asperger, A., and Deininger, S. O. (2004) Comparison of laser-induced dissociation and high-energy collision-induced dissociation using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOE/TOF) for peptide and protein identification. Rapid Commun. Mass Spectrom. 18, 2093-2105. 

Additional program(s) to validate the MS/MS-based peptide and protein identifications, to summarize results including MS/MS spectral counts (Scaffold, Proteome Software, Portland, OR), and to create aligned extracted-ion chromatograms (XICs, Sieve , Thermo), and so on. 

Scaffold (Proteome Software) is used to validate the MS/MS-based peptide and protein identifications. Peptide identifications are accepted if they could be estabhshed at greater than 95% probability as specified by the PeptideProphet algorithm [11]. Protein identifications, where protein probabilities are assigned by the ProteinProphet algorithm [12], are accepted if they can be established at greater than 99.0% probability and contain at least two identified peptides. 

Nesvizhskii AL A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics. J Proteomics. 2010 73 2092-123. doi 10.1016/j.jprot.2010.08.009. 

Shteynbeig D, Deutsch EW, Lam H, et al. iProphet multi-level integrative analysis of shotgun proteomic data improves peptide and protein identification rates and error estimates. Mol Cell Proteomics. 2011 10(12) M111.007690. doi 10.1074/mcp.M111.007690. 

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