Top Down Mass Spectrometry of <60-kDa Proteins from Methanosarcina acetivorans Using Quadrupole FTMS with Automated Octopole Collisionally Activated Dissociation

doi:10.1074/mcp.M500219-MCP200

Top Down Mass Spectrometry of <60-kDa Proteins from Methanosarcina acetivorans Using Quadrupole FTMS with Automated Octopole Collisionally Activated Dissociation*S

From the Departments of ^‡Chemistry and ^§Microbiology, University of Illinois, Urbana, Illinois 61801

¶To whom correspondence should be addressed: Dept. of Chemistry, 53 RAL, University of Illinois, 600. S. Mathews, Urbana, IL 61801. Tel.: 217-244-3927; Fax: 217-244-8068; E-mail: kelleher{at}scs.uiuc.edu

Abstract

A fragmentation geometry based upon axial acceleration of m/z-selected protein ions into a linear octopole ion trap allowed simultaneous production and external accumulation of fragment ions prior to m/z measurement in a FT mass spectrometer. Improved dynamic range resulting from this octopole collisionally activated dissociation resulted in a 2.5× increase in experimental throughput and a 2× increase in fragment ion matches to gene products identified and characterized in the top down fashion. The acceleration voltage for optimal fragmentation has a m/z and mass dependence, knowledge of which facilitated an automated platform for top down MS/MS on a quadrupole FT hybrid mass spectrometer. Controlled by improved software for data acquisition (e.g. using dynamic exclusion of previously identified species), automated octopole collisionally activated dissociation of samples fractionated using chromatofocusing and reversed-phase liquid chromatography achieved a significant increase in protein identification rate versus previous benchmarks. Also a batch analysis version of ProSight PTM facilitated probability-based identification of intact proteins obtained in a higher throughput fashion. In total, 101 unique proteins (5–59 kDa) were identified from whole cell lysates of Methanosarcina acetivorans grown anaerobically, including the characterization of several mispredicted start sites and biologically relevant mass discrepancies.

Footnotes

Published, MCP Papers in Press, October 18, 2005, DOI 10.1074/mcp.M500219-MCP200
↵1 The abbreviations used are: Q, quadrupole; CAD, collisionally activated dissociation; OCAD, octopole collisionally activated dissociation; MSAD, multipole storage-assisted dissociation; 2D, two-dimensional; ALS, acid-labile surfactant; RP, reversed-phase; PTM, post-translational modification; rf, radio frequency; S/N, signal to noise ratio; dc, direct current; PF, protein fractionation.
↵* This work was supported by the Packard Foundation and National Science Foundation Grant CHE-0134953.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
- Received July 18, 2005.
- Revision received October 17, 2005.