Delayed Fragmentation and Optimized Isolation Width Settings for Improvement of Protein Identification and Accuracy of Isobaric Mass Tag Quantification on Orbitrap-Type Mass Spectrometers

Abstract

Fragmentation of multiple peptides in a single tandem mass scan impairs accuracy of isobaric mass tag based quantification. Consequently, practitioners aim at fragmenting peptide ions with the highest possible purity without compromising on sensitivity and coverage achieved in the experiment. Here we report the first systematic study optimizing delayed fragmentation options on Orbitrap instruments. We demonstrate that by delaying peptide fragmentation to occur closer to the apex of the chromatographic peak in liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments cofragmentation is reduced by 2-fold and peptides are fragmented with 2.8-fold better signal-to-noise ratios. This results in significantly improved accuracy of isobaric mass tag quantification. Further, we measured cofragmentation dependence on isolation width. In comparison to Orbitrap XL instruments the reduced space charging in the Orbitrap Velos enables isolation widths as narrow as 1 Th without impairing coverage, thus substantially reducing cofragmentation. When delayed peptide fragmentation and narrow isolation width settings were both applied, cofragmentation-induced ratio compression could be reduced by 32% on a log2 scale under otherwise identical conditions.

Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase

“…Peptide fold changes were corrected for isotope purity as described and adjusted for interference caused by coeluting nearly isobaric peaks as estimated by the signal-to-interference measure (36). Protein quantification was achieved using a sum-based bootstrap algorithm (37). Apparent dissociation constants were determined by taking into account the protein depletion by the beads as described (37).…”

Evaluation and Improvement of Quantification Accuracy in Isobaric Mass Tag-Based Protein Quantification Experiments

“…discarding spectra with especially high levels of co-fragmentation reflected by high S2I values or Precursor abundance-to-threshold ratios (P2T) 14 .…”

“…This includes an algorithm, which detects co-isolated peptide signals in the MS1 spectrum and subsequently estimates and adjusts for co-fragmentation in the MS2 spectrum 13 . In another study, extensive fractionation, narrowing the precursor ion isolation width as well as delaying peptide fragmentation to occur closer to the apex of the chromatographic peak, have been demonstrated to lessen co-fragmentation and ratio compression 14 .…”

Functional interdependence of BRD4 and DOT1L in MLL leukemia

“…Reporter-ion intensities were read from raw data and multiplied with ion accumulation times (in units of milliseconds) to yield a measure proportional to the number of ions; this measure is referred to as ion area 57 . Spectra matches to peptides were filtered according to the following criteria: mascot ion score >15, signal-to-background of the precursor ion >4 and signal-to-interference >0.…”

“…Fold changes were corrected for isotope purity as described and adjusted for interference caused by coeluting nearly isobaric peaks, as estimated by the signal-to-interference measure 59 . Protein quantification was derived from individual spectra matching distinct peptides with a sum-based bootstrap algorithm; 95% confidence intervals were calculated for all protein fold-changes that were quantified with more than three spectra 57 .…”