Top down mass spectrometry of < 60-kDa proteins from Methanosarcina acetivorans using quadrupole FRMS with automated octopole collisionally activated dissociation.
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.
Patrie
Steve M
SM
Department of Chemistry, University of Illinois, Urbana, 61801, USA.
Ferguson
Jonathan T
JT
Robinson
Dana E
DE
Whipple
Dave
D
Rother
Michael
M
Metcalf
William W
WW
Kelleher
Neil L
NL
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D019843:Archaeal Proteins / Q000032:analysis* / Q000737:chemistry; D002853:Chromatography, Liquid; D030562:Databases, Protein; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D017020:Methanosarcina / Q000737:chemistry* / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D020539:Sequence Analysis, Protein / Q000295:instrumentation / Q000379:methods; D017386:Sequence Homology, Amino Acid; D012984:Software; D017550:Spectroscopy, Fourier Transform Infrared
D019843:Archaeal Proteins