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Proteomics on an Orbitrap Benchtop Mass Spectrometer Using All-ion Fragmentation

Open access
Geiger, Tamar, Cox, Juergen, Mann, Matthias·2010·Molecular & Cellular Proteomics

Abstract

The orbitrap mass analyzer combines high sensitivity, high resolution, and high mass accuracy in a compact format. In proteomics applications, it is used in a hybrid configuration with a linear ion trap (LTQ-Orbitrap) where the linear trap quadrupole (LTQ) accumulates, isolates, and fragments peptide ions. Alternatively, isolated ions can be fragmented by higher energy collisional dissociation. A recently introduced stand-alone orbitrap analyzer (Exactive) also features a higher energy collisional dissociation cell but cannot isolate ions. Here we report that this instrument can efficiently characterize protein mixtures by alternating MS and "all-ion fragmentation" (AIF) MS/MS scans in a manner similar to that previously described for quadrupole time-of-flight instruments. We applied the peak recognition algorithms of the MaxQuant software at both the precursor and product ion levels. Assignment of fragment ions to co-eluting precursor ions was facilitated by high resolution (100,000 at m/z 200) and high mass accuracy. For efficient fragmentation of different mass precursors, we implemented a stepped collision energy procedure with cumulative MS readout. AIF on the Exactive identified 45 of 48 proteins in an equimolar protein standard mixture and all of them when using a small database. The technique also identified proteins with more than 100-fold abundance differences in a high dynamic range standard. When applied to protein identification in gel slices, AIF unambiguously characterized an immunoprecipitated protein that was barely visible by Coomassie staining and quantified it relative to contaminating proteins. AIF on a benchtop orbitrap instrument is therefore an attractive technology for a wide range of proteomics analyses.

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Metadata

Authors
Geiger, T., Cox, J., & Mann, M.
Venue
Molecular & Cellular Proteomics
Publisher
Elsevier BV
Published
2010-10
Volume
9
Issue
10
Pages
2252-2261
ISSN
1535-9476 (ISSN-L)
OpenAlex
W2143340505
Cited by
254
References
8
FWCI
9.40
Type
journal article
Language
en
Open access
hybrid

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