Peptide protein identification

The greater amount of peptide/protein identification in the RP-RP experiment is likely related to higher peptide recovery from the first LC dimension. In addition, a... 

Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

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... 

Bemdt, P., Hobohm, U., and Langen, H. (1999). Reliable automatic protein identification from matrix-assisted laser desorption/ionization mass spectrometric peptide fingerprints. Electrophoresis 20, 3521-3526. 

Wienkoop, S., Glinski, M., Tanaka, N., Tolstikov, V.V., Fiehn, O., Weckwerth, W. (2004). Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins. Rapid Commun. Mass Spectrom. 18, 643-650. 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

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 combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

This chapter has presented several comprehensive 2DLC approaches combining a first-dimension IEX separation and a second-dimension RP separation for the analysis of complex protein mixtures typical in proteomics studies. Online ESI-TOF/MS detection provided sensitive detection and valuable qualitative information (MW) for proteins eluting from the MDLC system. Coordinated fraction collection and subsequent MS analysis of peptides produced by proteolysis of the fractions provided in-depth information on protein identification and a mechanism... 

Janini, G.M., Chan, K.C., Conrads, T.P., Issaq, H.J., Veenstra, T.D. (2004). Two-dimensional liquid chromatography-capillary zone electrophoresis—sheathless electrospray ionization-mass spectrometry evaluation for peptide analysis and protein identification. Electrophoresis 25, 1973-1980. 

All samples were digested with trypsin and analyzed by cIEF in the first dimension followed by LC-MS/MS as described above. Samples were analyzed in duplicate. Sequence searching was performed using OMSSA. Analysis of the soluble fraction yielded a total of 2856 identified proteins, while the insoluble fraction yielded 3227 proteins. Combined, the fresh-frozen sample yielded 3902 protein identifications. The FFPE portion yielded 2845 protein identifications from 14,178 distinct tryptic peptide sequences, on a par with the fresh-frozen soluble fraction. Combining all identifications gave 4145 proteins. While, the soluble fraction and the FFPE extraction yielded similar numbers of protein identification, both found 25% of their respective protein set uniquely (Fig. 20.4). 

Three human FFPE liver cases were analyzed and each yielded just under 20,000 distinct peptide sequence identifications and just over 3000 protein... 

This procedure is called Peptide Mass Mapping (PMM), which is defined as a means of protein identification by comparing observed masses (m/z values) with predicted masses of digested proteins contained in a database. 

Chemical, thermal, or enzymatic treatments are required to obtain analysable samples. Two typical methods used to achieve the hydrolysis of peptidic bonds are enzymatic and chemical catalysis [73]. The reaction times for enzymatic hydrolysis are long and typically lie in the range of 4 8 h [47]. Additionally, they demand purification procedures to get rid of the excess enzyme that could interfere in the protein identification. Due to these drawbacks, this method of hydrolysis finds limited use in the conservation science field. 

J. Gao, J.D. Xu, L.E. Locascio, and C.S. Lee, Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal. Chem. 73, 2648-2655 (2001). 

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