Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Ion Mobility MS
Ion mobility separation (IM/IMS) is of value to the analysis of post-translationally modified peptides, particularly of positional isomers. So-called “conventional IMS” measures the absolute mobility at moderate electric field strength. Field asymmetric waveform IMS (FAIMS, also known as differential mobility separation) is based on the difference between absolute mobility values at high and low electric field strengths. UltraFAIMS and SelexION devices are variants of the FAIMS device. A range of mass analyzers can be coupled to IMS including TOF, linear quadrupole, ion trap, and orbitrap instrumentation.
The introduction of differential IMS, based on gas-phase collisional crosssection (CCS), reflects the ion topology of ions and provides an orthogonal measurement to m/z. The use of IMS provides an additional ion characteristic, termed drift time (order of milliseconds), to combine with retention time, m/z, and intensity information, providing another dimension of separation to extend the resolving power of HPLC by increasing selectivity and peak capacity [70, 71]. The combination of retention and drift time information improves the accuracy of assignment of CID fragments to precursors as demonstrated for TWIMS, by resolving chimeric MS2 spectra of coeluting peptides and reduction of chemical noise. [72, 73]. Additional benefits accrue for improved quantitative performance of isobaric tag-based quantitation by reducing coisolation of mixed precursors of same m/z value relative to DDA . IMS-DDA has been applied to protein acetylation research using TMT quantitative chemoproteomic analysis to identify cellular proteins binding immobilized HDAC inhibitors .
The combination of TWIMS with DIA provides high-definition MSE . The introduction of ETD capability offers complementary data to IMS-CID data for PTM assignment and quantification . IMS post-ETD enables the assignment of fragments to a specific charge-reduced state of the precursor, a particular benefit for top-down analysis of proteins . Furthermore, combination of IMS-CID and ETD-MS generates multistage MS (MS„) data, which can aid PTM assignment . IMS is an appropriate resolving tool for PTMs where the PTM induces a change in the CCS of the analyte under investigation. FAIMS spectra have been used to resolve methylation and acetylation site localization variants at the bottom-up and middle-down levels .
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