False-Positive Glycopeptide Identification via In-FAIMS Fragmentation

Abstract

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates glycopeptides in the gas phase prior to mass spectrometry (MS) analysis, thus offering the potential to analyze glycopeptides without prior enrichment. Several studies have demonstrated the ability of FAIMS to enhance glycopeptide detection but have primarily focused on N-glycosylation. Here, we evaluated FAIMS for O-glycoprotein and mucin-domain glycoprotein analysis using samples of varying complexity. We demonstrated that FAIMS was useful in increasingly complex samples as it allowed for the identification of more glycosylated species. However, during our analyses, we observed a phenomenon called “in FAIMS fragmentation” (IFF) akin to in source fragmentation but occurring during FAIMS separation. FAIMS experiments showed a 2- to 5-fold increase in spectral matches from IFF compared with control experiments. These results were also replicated in previously published data, indicating that this is likely a systemic occurrence when using FAIMS. Our study highlights that although there are potential benefits to using FAIMS separation, caution must be exercised in data analysis because of prevalent IFF, which may limit its applicability in the broader field of O-glycoproteomics.

A Tutorial Review of Labeling Methods in Mass Spectrometry-Based Quantitative Proteomics

“…FAIMS has been shown to significantly enhance the dynamic range and accuracy of quantitative measurements without sacrificing protein identifications. − FAIMS-SPS-MS3 method was reported to have the highest quantitative accuracy followed by SPS-MS3, FAIMS-HRMS2, and HRMS2. , Recently, Fang et al highlighted the utility of FAIMS in improving the accuracy of N-glycopeptide quantification, showcasing its versatility in post-translational modifications (PTM) analysis . However, Rangel-Angarita et al also reported that in-FAIMS fragmentation can lead to false positive detection of glycopeptides, especially when high compensation voltage (CV) values was applied …”

Glycoproteomics: Charting new territory in mass spectrometry and glycobiology

“…2,5,29 Enrichment is commonly used to lessen effects of ion suppression; by selectively isolating glycopeptides from unmodified species, their detection is greatly improved. 29,33 An exorbitant number of enrichments exist, including (but not limited to) hydrophilic interaction chromatography (HILIC) and lectin-based affinity chromatography, as well as those that leverage chemical biology tools. [34][35][36] However, each of these methods is biased for subsets of the glycoproteome, and a universal enrichment tool does not yet exist.…”

Experimentally Determined Diagnostic Ions for Identification of Peptide Glycotopes

“…The location of these identified glycotopes cannot be accurately determined without a corresponding Y1 glycopeptide fragment, which provides information on the glycan attachment and localization, or through analysis of the peptide sequence to identify the site of attachment. To add to the dangers of potentially incorrect assignment of glycotopes, the inherent lability of glycans in a mass spectrometer can lead to MS1 level fragmentation, resulting in misinterpretation of results. − This can be further confounded by reported migration of monosaccharides and other modifications within a glycan. − It is also important to consider the current knowledge of glycan motifs to be incomplete, and novel motifs may in the future be found that have the same fragment masses as the ions compiled in this piece. Care must be taken in the use of glycotopes for assigning complete structural features without additional glycotope information, for example, assigning an oligomannose N-glycan after observing a Hex ion ( m / z 163.0601) without additional information where this ion could be a product of a Gal capped N-glycan, or an O-glycan.…”