Proteomics on an Orbitrap benchtop mass spectrometer using all ion fragmentation
Mol Cell Proteomics. 2010 Sep 10. [Epub ahead of print]
Geiger T, Cox J, Mann M.
The orbitrap mass analyzer combines high sensitivity, high resolution and high mass accuracy in a compact format. In proteomic applications it is used in a hybrid configuration with a linear ion trap (LTQ-Orbitrap), where the LTQ accumulates, isolates and fragments peptide ions. Alternatively, isolated ions can be fragmented by higher energy collisional dissociation (HCD). A recently introduced stand-alone orbitrap analyzer (Exactive) also features an HCD 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 hundred-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 proteomic analyses.