文摘
Tandem mass spectrometry (MS/MS) plays an importantrole in the unambiguous identification and structuralelucidation of biomolecules. In contrast to conventionalMS/MS approaches for protein identification where anindividual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation(IRMPD) or multiple frequency sustained off-resonanceirradiation (SORI) collisionally induced dissociation (CID).The high mass measurement accuracy and resolution ofFTICR combined with knowledge of peptide dissociationpathways allows the fragments arising from several different parent ions to be assigned. Herein we report theapplication of multiplexed-MS/MS coupled with on-lineseparations for the identification of peptides present incomplex mixtures (i.e., whole cell lysate digests). Softwarewas developed to enable "on-the-fly" data-dependent peakselection of a subset of polypeptides from each FTICR MSacquisition. In the subsequent MS/MS acquisitions,several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. Theutility of this approach has been demonstrated using abovine serum albumin tryptic digest separated by capillaryLC where multiple peptides were readily identified insingle MS/MS acquisitions. We also present initial resultsfrom multiplexed-MS/MS analysis of a D. radioduranswhole cell digest to illustrate the utility of this approachfor high-throughput analysis of a bacterial proteome.