A Rapid Approach to the Identification and Characterization of Tryptic Peptides Using High Linear Velocity Nanobore UPLC Separations Coupled with ESI MS/MS

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Iain Campuzano, Ian Ross, Charalampos-Philip Iossifidis, Thérèse McKenna, Jim Langridge
ProteomeLux 2006 October 11 - 14 Luxembourg; Proteome Analysis in Systems Biology December 7 - 8 Antwerp, Belgium; ABRF The Association of Biomolecular Resource Facilities, March 31 - April 3, Tampa, Florida; BSBMB 195th Meeting of the Belgian Society of Biochemistry and Molecular Biology; May 15, 2007, Bruxelles; Hellenics Proteomic Society May 23 - 25, Crete, Greece
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Mass spectrometry has now firmly established itself as the primary technique for identifying proteins due to its unparalleled speed, sensitivity and specificity. Strategies can involve either analysis of the intact protein, or more commonly digestion of the protein using a specific protease that cleaves at predictable residues along the peptide backbone. This provides smaller stretches of peptide sequence that are more amenable to analysis via mass spectrometry. When coupled with protein level pre-fractionation strategies, thus reducing the complexity of the protein mixture, this approach has proven highly successful in comprehensive protein identification and characterisation. A common approach to protein pre-fractionation is the use of 1 dimensional PAGE, coupled with LC-MS/MS. The downside of this approach is the number of gel samples, or fractions, to be analysed by the LC-MS/MS system. With typical analytical HPLC run times of 45 minutes to 1 hour the amount of time required to analyses one top level sample can be prohibitive.

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