Sub-part-per-million precursor and product mass accuracy for high-throughput proteomics on an electron transfer dissociation-enabled orbitrap mass spectrometer

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.

Fig. 1.

Schematic depiction of fluoranthene cation internal calibrant scan event. First, analyte cations (blue) are injected through the source region into the linear ion trap, isolated and fragmented (in the case of MS/MS), and then transferred to the c-trap. Next, calibrant fluoranthene cations (red) from the CI source are transferred through a multipole and the collision cell into the c-trap. Finally, all the ions are transferred together to the orbitrap for high-resolution and high-mass accuracy analysis.

Fig. 2.

Mass chromatograms and single scan spectra from replicate LC-MS/MS run 30. Although the analyte signal varies greatly over the chromatographic gradient for both MS¹ (a) and MS² (c) scan events, the internal calibrant signal remains consistent and robust in both MS¹ (b) and MS² (d) spectra.

Fig. 3.

Mass error distributions for 1934 precursors (a) and ∼24,000 products (b) detected in orbitrap during replicate LC-MS/MS run 60. The data were analyzed with standard external mass calibration (dark gray), linear internal mass recalibration (light gray), and proportional internal mass recalibration (medium gray). This run started about 134 h (∼5.5 days) after external mass calibration. Both internal mass recalibration strategies show improvement in the center and width of the distribution, although proportional internal mass recalibration is superior.

Fig. 4.

Mass calibration drift for precursors (a) and products (b) over 60 replicate LC-MS/MS runs performed after external mass calibration. A Gaussian fit was applied to the mass error distribution for each run before (red) and after linear (green) and proportional (blue) internal mass recalibration; error bars represent ±1 S.D. For both MS¹ and MS² scans, proportional internal mass recalibration virtually eliminated drift and reduced the width of the distributions.

Fig. 5.

Mass error distributions for ∼120,000 precursors (a) and 1.6 million products (b) measured over 60 replicate LC-MS/MS runs. The data were treated with external mass calibration (dark gray), linear internal mass recalibration (light gray), and proportional internal mass recalibration (medium gray). Although the center of the mass error distributions did not improve much after mass recalibration, drift correction led to substantially narrower peaks.