Sub-part-per-million precursor and product mass accuracy for high-throughput proteomics on an electron transfer dissociation-enabled orbitrap mass spectrometer.
We demonstrate a new approach for internal mass calibration on an electron transfer dissociation-enabled linear ion trap-orbitrap hybrid mass spectrometer. Fluoranthene cations, a byproduct of the reaction used for generation of electron transfer dissociation reagent anions, are co-injected with the analyte cations in all orbitrap mass analysis events. The fluoranthene cations serve as a robust internal calibrant with minimal impact on scan time (<20 ms) or spectral quality. Following external mass calibration, 60 replicate LC-MS/MS runs of a complex peptide mixture were collected over the course of approximately 136 h (almost 6 days). Using only standard external mass calibration, the mass accuracy for a typical analysis was -3.31 +/- 0.93 ppm (sigma) for precursors and -2.32 +/- 0.89 ppm for products. After application of internal recalibration, mass accuracy improved to +0.77 +/- 0.71 ppm for precursors and +0.17 +/- 0.67 ppm for products. When all 60 replicate runs were analyzed together without internal mass recalibration, the mass accuracy was -1.23 +/- 1.54 ppm for precursors and -0.18 +/- 1.42 ppm for products, nearly a 2-fold drop in precision relative to an individual run. After internal mass recalibration, this improved to +0.80 +/- 0.70 ppm for precursors and +0.16 +/- 0.67 ppm for products, roughly equivalent to that obtained in a single run, demonstrating a near complete elimination of mass calibration drift.
Wenger
Craig D
CD
Department Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
McAlister
Graeme C
GC
Xia
Qiangwei
Q
Coon
Joshua J
JJ
D002138:Calibration; D002460:Cell Line; D002853:Chromatography, Liquid; D004583:Electrons; D005449:Fluorenes; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D005449:Fluorenes; C007738:fluoranthene