Large-Scale Collision Cross-Section Profiling on a Traveling Wave Ion Mobility Mass Spectrometer

Abstract

Ion mobility (IM) is a gas-phase electrophoretic method that separates ions according to charge and ion-neutral collision cross-section (CCS). Herein, we attempt to apply a travelling wave (TW) IM polyalanine calibration method to shotgun proteomics and create a large peptide CCS database. Mass spectrometry methods that utilize IM, such as HDMSE, often use high transmission voltages for sensitive analysis. However, polyalanine calibration has only been demonstrated with low voltage transmission used to prevent gas-phase activation. If polyalanine ions change conformation under higher transmission voltages used for HDMSE, the calibration may no longer be valid. Thus, we aimed to characterize the accuracy of calibration and CCS measurement under high transmission voltages on a TW IM instrument using the polyalanine calibration method and found that the additional error was not significant. We also evaluated the potential error introduced by liquid chromatography (LC)-HDMSE analysis, and found it to be insignificant as well, validating the calibration method. Finally, we demonstrated the utility of building a large-population peptide CCS database by investigating the effects of terminal lysine position, via LysC or LysN digestion, on the formation of two structural sub-families formed by triply charged ions.

Ion Mobility Collision Cross Section Compendium

“…, hydrocarbons and metabolites; 6%) representing the remainder of values. The focus on peptide and protein work can be rationalized as being a result of continued efforts for adapting ion mobility technologies to proteomics workflows, 127–129 but also a practical consequence of both the ease of generating large pools of peptides derived from enzymatic digestion 130 and the fact that the structural and charge-state heterogeneity of proteins necessitates the reporting of many CCS values for a single protein. 131–134 …”

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

“…Like other TWIMS calibration strategies [25,30,33,50,[53][54][55], the procedure used here relies on drift tube reference data Ω ref measured in He. Calibrated TWIMS Ω values therefore represent Beffective^He collision cross sections, although TWIMS uses N 2 as IMS gas [33,60].…”

“…Cone voltages greater than 100 V start to cause calibrant breakdown by CID (Supporting Figure S1) [67], thereby identifying the upper limit of the useful parameter space. The fact that the TWIMS profiles of many calibrant ions depend on the level of collisional activation represents a problem [54,60]. A reliable calibration requires TWIMS and drift tube reference data to be acquired using the same level of activation.…”

“…Hence, TWIMS data have to be calibrated using drift tube reference values Ω ref obtained on the basis of Equation 1 [25,30,33,50,[53][54][55]. A calibrant mix comprising Ubq, Cyt, and aMb in denaturing solution is commonly used for this purpose [30,33].…”

Electron transfer dissociation of sodium cationized polyesters: Reaction time effects and combination with collisional activation and ion mobility separation

“…Calibrants were the [M+H] + ions of polyalanine oligomers whose˝values had been previously established by standard DC-drift tube IM-MS experiments [36,37] Several studies have shown that CCSs estimated by the described procedure, i.e. from calibrated T-wave devices, and CCSs measured with drift tube ion mobility instrumentation differ by less than ±3% [33,35,[38][39][40][41]. When this uncertainty is combined with the reproducibility of our drift time measurements (±<1%) and the error reported for the calibrant CCSs (±<1%), the experimental errors for the CCS values reported in this study fall within ±3-4%.…”