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.
Highlights
The orbitrap mass analyzer combines high sensitivity, high resolution, and high mass accuracy in a compact format
The Exactive benchtop instrument consists of a single orbitrap mass analyzer and no linear trap quadrupole (LTQ); peptides cannot be isolated for fragmentation (Fig. 1A)
We wished to examine the applicability of this instrument to proteomics by basing peptide identification on higher energy collisional dissociation (HCD) fragmentation without precursor peptide selection, which is termed all-ion fragmentation” (AIF)
Summary
BSA Sample Preparation—Bovine serum albumin fraction V (Sigma) was solubilized in 8 M urea solution (6 M urea ϩ 2 M thiourea in 10 mM HEPES, pH 8) to a concentration of 350 g/ml. The protein sample was split, and 20% was used for Western blot, and 80% was used for further MS analysis In both cases, proteins were separated on a NuPAGE Bis-Tris gel (Invitrogen). The Exactive mass spectrometer was operated in positive ion mode with alternating MS scans of the precursor ions and AIF scans in which the peptides were fragmented by HCD. Both scan types were performed with 100,000 resolution (at m/z 200) with each scan taking 1 s, and the maximal fill time was set to 1 s as well. To avoid apparent misidentifications that are only due to protein name discrepancies [4], we manually examined gene names and UniProt ids and compared them to the list of proteins given by the manufacturer
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