High-Throughput Peptide Identification from Protein Digests Using Data-Dependent Multiplexed Tandem FTICR Mass Spectrometry Coupled with Capillary Liquid Chromatography

Abstract

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual 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-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

“…The simulation data indicate that peptides can be confidently identified from multiplexed data from simultaneous dissociation of ten peptides, consistently with our pre-vious experimental studies [7][8][9]. We further support those results by experimental data using a BSA tryptic digest analyzed by LC/ESI-MS.…”

“…The instruments and the experimental sequences for multiplexed MS/MS have been described in detail elsewhere [7,8,11]. BSA was purchased from Sigma (St. Louis, MO, USA) and digested overnight (at 377C) using modified trypsin (Promega, Madison, WI, USA).…”

“…Multiplexed MS/MS provides a possible basis for faster and therefore more sensitive peptide identification in LC-MS/MS experiments [7,8]. In this approach, which relies upon high mass accuracy MS data, a set of parent species is dissociated and measured in a single mass spectrum (i.e., in exactly the same manner that a single parent species is conventionally studied), providing both enhanced sensitivity and a gain in throughput directly proportional to the number of species that may be simultaneously addressed [8].…”

“…In this approach, which relies upon high mass accuracy MS data, a set of parent species is dissociated and measured in a single mass spectrum (i.e., in exactly the same manner that a single parent species is conventionally studied), providing both enhanced sensitivity and a gain in throughput directly proportional to the number of species that may be simultaneously addressed [8]. As an initial proof of principle, a set of seven peptides was dissociated in a single experiment and unambiguously identified among all the possible tryptic fragments of the total predicted protein complement of the Caenorhabditis elegans genome [7].…”

“…As an initial proof of principle, a set of seven peptides was dissociated in a single experiment and unambiguously identified among all the possible tryptic fragments of the total predicted protein complement of the Caenorhabditis elegans genome [7]. In a subsequent report, the coupling of multiplexed MS/MS with on-line LC was demonstrated [8]. The approach was successfully applied to the identification of a protein from the SWISS-PROT database, and for protein identification from a complex bacterial proteome sample.…”