Abstract
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.
Highlights
We demonstrate a new approach for internal mass calibration on an electron transfer dissociation-enabled linear ion trap-orbitrap hybrid mass spectrometer
The chemical ionization (CI) reaction that produces radical fluoranthene anions for ETD simultaneously produces a robust flux of fluoranthene cations
Mass accuracy was something of an afterthought throughout the early days of shotgun proteomics
Summary
Instrumentation—All data were collected using an ETD-enabled LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The fluoranthene cation internal calibrant peak at m/z 202.07770 was introduced throughout data collection. ETD spectral pre-processing was performed on all MS/MS spectra to remove interfering peaks from unreacted precursor, charge-reduced precursors (ETnoD), and neutral losses from ETnoD. Internal mass recalibration was performed post-acquisition in DTA Generator for both MS1 and MS2 spectra based on the fluoranthene radical cation internal calibrant at a theoretical m/z of 202.07770 using either a linear or proportional m/z correction. The peak selected for isolation was located in the MS1 spectrum and converted to neutral mass using the known precursor charge state This mass was corrected for isotopic shift by subtracting an integer multiple of 1.003355 Da (13C Ϫ 12C) to yield the mass closest to the theoretical neutral monoisotopic mass. These parameters for the center and width of the distribution can be thought of as the accuracy and precision, respectively, of the mass measurement
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