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

Citations Over TimeTop 10% of 2011 papers

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.

Related Papers

• → Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos(2011)307 cited
• → High Precision Quantitative Proteomics Using iTRAQ on an LTQ Orbitrap: A New Mass Spectrometric Method Combining the Benefits of All(2009)150 cited
• → Isobaric protein and peptide quantification: perspectives and issues(2010)142 cited
• → Increased N,N-Dimethyl Leucine Isobaric Tag Multiplexing by a Combined Precursor Isotopic Labeling and Isobaric Tagging Approach(2018)40 cited
• → Differential quantification of isobaric phosphopeptides using data-independent acquisition mass spectrometry(2016)13 cited