Journal
ANALYTICAL CHEMISTRY
Volume 83, Issue 23, Pages 8959-8967Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac201760x
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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 cofragmentation-induced ratio compression could be reduced by 32% on a log2 scale under width settings were both applied, otherwise identical conditions.
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