Top down mass spectrometry of <60-kDa proteins from Methanosarcina acetivorans using quadrupole FTMS with automated octopole collisionally activated dissociation

Steven M. Patrie, Jonathan T. Ferguson, Dana E. Robinson, Dave Whipple, Michael Rother, William W. Metcalf, Neil L. Kelleher

Research output: Contribution to journalArticle

54 Citations (Scopus)

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 2.5x increase in experimental throughput and a 2x 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.

Original languageEnglish (US)
Pages (from-to)14-25
Number of pages12
JournalMolecular and Cellular Proteomics
Volume5
Issue number1
DOIs
StatePublished - Jan 2006

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Methanosarcina
Mass spectrometry
Mass Spectrometry
Ions
Mass spectrometers
Proteins
Throughput
Pulse time modulation
Benchmarking
Liquid chromatography
Reverse-Phase Chromatography
Data acquisition
Software
Genes
Geometry
Electric potential

ASJC Scopus subject areas

  • Biochemistry

Cite this

Top down mass spectrometry of <60-kDa proteins from Methanosarcina acetivorans using quadrupole FTMS with automated octopole collisionally activated dissociation. / Patrie, Steven M.; Ferguson, Jonathan T.; Robinson, Dana E.; Whipple, Dave; Rother, Michael; Metcalf, William W.; Kelleher, Neil L.

In: Molecular and Cellular Proteomics, Vol. 5, No. 1, 01.2006, p. 14-25.

Research output: Contribution to journalArticle

Patrie, Steven M. ; Ferguson, Jonathan T. ; Robinson, Dana E. ; Whipple, Dave ; Rother, Michael ; Metcalf, William W. ; Kelleher, Neil L. / Top down mass spectrometry of <60-kDa proteins from Methanosarcina acetivorans using quadrupole FTMS with automated octopole collisionally activated dissociation. In: Molecular and Cellular Proteomics. 2006 ; Vol. 5, No. 1. pp. 14-25.
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