Additional Precursor Purification in Isobaric Mass Tagging Experiments by Traveling Wave Ion Mobility Separation (TWIMS)

Abstract

Despite the increasing popularity of data-independent acquisition workflows, data-dependent acquisition (DDA) is still the prevalent method of LC-MS-based proteomics. DDA is the basis of isobaric mass tagging technique, a powerful MS2 quantification strategy that allows coanalysis of up to 10 proteomics samples. A well-documented limitation of DDA, however, is precursor coselection, whereby a target peptide is coisolated with other ions for fragmentation. Here, we investigated if additional peptide purification by traveling wave ion mobility separation (TWIMS) can reduce precursor contamination using a mixture of Saccharomyces cerevisiae and HeLa proteomes. In accordance with previous reports on FAIMS-Orbitrap instruments, we find that TWIMS provides a remarkable improvement (on average 2.85 times) in the signal-to-noise ratio for sequence ions. We also report that TWIMS reduces reporter ions contamination by around one-third (to 14-15% contamination) and even further (to 6-9%) when combined with a narrowed quadrupole isolation window. We discuss challenges associated with applying TWIMS purification to isobaric mass tagging experiments, including correlation between ion m/z and drift time, which means that coselected peptides are expected to have similar mobility. We also demonstrate that labeling results in peptides having more uniform m/z and drift time distributions than observed for unlabeled peptides. Data are available via ProteomeXchange with identifier PXD001047.

Ion Mobility Tandem Mass Spectrometry Enhances Performance of Bottom-up Proteomics

“…A recent report has shown that TWIMS can improve the quantitative performance of isobaric mass tagging experiments (31,32). Notably, the ion mobility separation of peptide precursor ions reduced the well described issue of co-isolation of peptides and the concomitant decrease in accuracy and precision of TMT quantification (33).…”

Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-Based Quantitation in Proteomic LC/MS/MS

“…However, in such experiments, the advantages of isobaric tags cannot be exploited, because the total analysis time is increased, and the throughput is reduced. Ion mobility spectrometry (IMS) is compatible with LC and MS and enables increased sensitivity and selectivity in MS and an overall increase in peak capacity without increasing the total analysis time. , So far, LC/IMS/MS experiments using traveling wave ion mobility spectroscopy (TWIMS) and electric field asymmetric ion mobility spectroscopy (FAIMS) − have demonstrated that it is possible to uncompress the ratio of isobaric tags in quantitative proteome analysis.…”

Quantitative Proteomics Using Isobaric Labeling: A Practical Guide

“…Fourth, additional precursor purification using ion mobility separation has the potential to reduce isobaric interference since ion mobility MS separates ions based on charge, size, and shape [72] , [73] , [74] . Using a TMT-based interference standard [75] , high-field asymmetric waveform ion mobility MS (FAIMS) improves the quantitation accuracy of multiplexed quantitative analyses using both high-resolution MS2 and SPS-MS3-based methods [74] .…”

“…Third, a triethyl ammonium bicarbonate (TEAB) buffer is recommended by the manufacturers of TMT for isobaric labeling. However, the use of 50 mM TEAB in TMT labeling will introduce unidentified and unwanted side reaction substances, such as single charged ions that MS detects with m/z values of 303.26, 317.26, 331.29, and 391.25 [67] , [72] . These substances cannot be removed by RPC desalting.…”