Separation of Peptide Isomers with Variant Modified Sites by High-Resolution Differential Ion Mobility Spectrometry

Abstract

Many proteins and proteolytic peptides incorporate the same post-translational modification (PTM) at different sites, creating multiple localization variants with different functions or activities that may coexist in cells. Current analytical methods based on liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS) are challenged by such isomers that often co-elute in LC and/or produce non-unique fragment ions. The application of ion mobility spectrometry (IMS) was explored, but success has been limited by insufficient resolution. We show that high-resolution differential ion mobility spectrometry (FAIMS) employing helium-rich gases can readily separate phosphopeptides with variant modification sites. Use of He/N 2 mixtures containing up to 74% He has allowed separating to >95% three monophosphorylated peptides of identical sequence. Similar separation was achieved at 50% He, using an elevated electric field. Bisphosphorylated isomers that differ in only one modification site were separated to the same extent. We anticipate FAIMS capabilities for such separations to extend to other PTMs.

A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements

“…Previous studies have reported the benefits of FAIMS to improve proteome coverage and to reduce the extent of co-fragmentation that impedes the identification of low-abundance peptide ions (28)(29)(30)(31)(32)(33). The separation capability of FAIMS also facilitates the resolution of isomeric peptides, including histone variants and isomeric phosphopeptides (34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). Furthermore, the reduction of peptide co-elution and co-fragmentation observed with FAIMS can significantly improve the accuracy and the comprehensiveness of multiplex proteomic analyses (45).…”

Pushing the Frontier of High-Definition Ion Mobility Spectrometry Using FAIMS

“…High-resolution FAIMS appears able to generally separate the localization variants for all PTMs baseline, as demonstrated for phosphorylation 24,30) (80 Da, Fig. 4), O-linked glycosylation (by N-acetyl-galactosamine, 221 Da), 31) acetylation (42 Da), 32) and the smallest additive PTM-methylation (14 Da).…”

“…In particular, MS/MS can tell apart only two of three or more localization isomers because all fragments of others (with either collision-induced or electron capture/transfer dissociation) are isobaric to those from a mixture of the first two. 24,27) Consequently, PTMs are often assigned to a protein region rather than a residue, 28) although variants with PTMs at nearby sites may have different and even opposite activities in vivo.…”

“…23) With the peak positions now employed to identify analytes, the reproducibility of measured CVs also becomes crucial. 24) While internal calibration helps, its utility is limited by the inevitably unequal K(E) dependences for different species. 24) Starting to address these issues, we have constructed a new CV supply with a more precise and less noisy output.…”

“…24) While internal calibration helps, its utility is limited by the inevitably unequal K(E) dependences for different species. 24) Starting to address these issues, we have constructed a new CV supply with a more precise and less noisy output. 23) This electronics has narrowed the features at highest resolving power by ∼40 mV or ∼1/3, pushing the maximum R (for multiply-charged peptides) into the ∼400-500 range.…”