Effect of Phosphorylation on the Collision Cross Sections of Peptide Ions in Ion Mobility Spectrometry

Abstract

The insertion of ion mobility spectrometry (IMS) between LC and MS can improve peptide identification in both proteomics and phosphoproteomics by providing structural information that is complementary to LC and MS, because IMS separates ions on the basis of differences in their shapes and charge states. However, it is necessary to know how phosphate groups affect the peptide collision cross sections (CCS) in order to accurately predict phosphopeptide CCS values and to maximize the usefulness of IMS. In this work, we systematically characterized the CCS values of 4,433 pairs of mono-phosphopeptide and corresponding unphosphorylated peptide ions using trapped ion mobility spectrometry (TIMS). Nearly one-third of the mono-phosphopeptide ions evaluated here showed smaller CCS values than their unphosphorylated counterparts, even though phosphorylation results in a mass increase of 80 Da. Significant changes of CCS upon phosphorylation occurred mainly in structurally extended peptides with large numbers of basic groups, possibly reflecting intramolecular interactions between phosphate and basic groups.

Rapid and in-depth coverage of the (phospho-)proteome with deep libraries and optimal window design for dia-PASEF

“…Our workflow is also twice as fast as currently employed high throughput screening strategies for cancer proteomics, while achieving greater proteome depth on cell lysate (63)(64)(65). So far, there have been only a few reports of the timsTOF principle on phosphoproteomics (28). Here, we show that this instrument is capable of in-depth phosphoproteomics with very high sensitivity.…”

“…So far, there have been only a few reports of the timsTOF principle on phosphoproteomics (28). Here, we show that this instrument is capable of in-depth phosphoproteomics with very high sensitivity.…”

“…On the Orbitrap MS platform, Olsen and co-workers recently demonstrated an efficient combination of fast chromatography runs with DIA, quantifying more than 13,000 phosphopeptides in very short (15 min) LC/MS runs from HeLa cells using the Spectronaut software (11). In a small scale study, Ishihama and co-workers showed that phosphopeptides analysis benefits from the additional ion mobility dimension in PASEF (28). For large-scale PTM studies, our optimized py_diAID acquisition schemes cover nearly all theoretical phosphopeptide precursors and quantified expected changes in the well-studied EGF-receptor signaling pathway with minimal time and sample consumption.…”

Rapid and In-Depth Coverage of the (Phospho-)Proteome With Deep Libraries and Optimal Window Design for dia-PASEF

“…On the Orbitrap MS platform, Olsen and co-workers recently demonstrated an efficient combination of fast chromatography runs with DIA, quantifying more than 13,000 phosphopeptides in very short (15 min) LC/MS runs from HeLa cells using the Spectronaut software (11). In a small scale study, Ishihama and co-workers showed that phosphopeptides analysis benefits from the additional ion mobility dimension in PASEF (29). For large-scale PTM studies, our optimized py_diAID acquisition schemes cover nearly all theoretical J o u r n a l P r e -p r o o f phosphopeptide precursors and quantified expected changes in the well-studied EGFreceptor signaling pathway with minimal time and sample consumption.…”

Ion mobility-resolved phosphoproteomics with dia-PASEF and short gradients

“…Intriguingly, phosphorylation can have a distinct influence on the gas phase conformation of a peptide as measured by its collisional cross section (CCS) 21 . Analysis of a large number of phosphopeptides revealed that their distribution in the ion mobility vs m/z space is shifted towards more compact conformations as compared with unmodified peptides, suggesting sequence-specific interactions of the phosphoryl group with basic amino acids [22][23] .…”