PXD041217

PXD041217 is an original dataset announced via ProteomeXchange.

Title	False positive glycopeptide identification through in-FAIMS glycan fragmentation.
Description	Glycosylation is an important post-translational modification characterized by extensive heterogeneity. While mass spectrometry (MS) is the premier technique to characterize glycoproteins, the study of intact glycopeptides presents challenges often not observed in the study of unmodified species. High-field asymmetric waveform ion mobility spectrometry (FAIMS) involves in-line gas-phase separation and offers the potential to analyze glycopeptides without prior enrichment. While FAIMS has been assessed for its use in glycoproteomics, most of these studies focus heavily or exclusively on N-glycoproteomics. Therefore, we evaluated FAIMS for O-glycoprotein and mucin analysis. We generated three samples: the mucin-domain glycoprotein podocalyxin, a recombinant glycoprotein mixture, and a WGA enriched human platelet sheddome. Although FAIMS allowed for the identification of new podocalyxin O-glycosites, it yielded less glycopeptide identifications and glycoforms. FAIMS seemed to be more useful in the increasingly complex samples, since we observed a higher number of identified glycosylated species. Because of the labile nature of glycosidic bonds, and presence of a buffer gas in FAIMS separation, we investigated the possibility of increased in-source fragmentation during FAIMS analysis. We compared total ion chromatograms of identifications eluting within a minute of identifications bearing larger glycan structures, albeit with the same peptide backbone. FAIMS experiments showed a 2-5-fold increase in spectral matches from in-FAIMS fragmentation compared to control experiments. These results were also replicated in other previously published data, indicating that this is a systematic behavior. In summary, our study highlights that, although there are potential benefits gained when using FAIMS separation, caution must be exercised when using FAIMS due to in-FAIMS fragmentation, which limits its applicability in the field of O-glycoproteomics.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:31:21.661.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Valentina Rangel Angarita
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	complex glycosylation
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2023-03-30 07:24:15	ID requested
1	2023-09-11 12:44:44	announced
⏵ 2	2023-11-14 08:31:22	announced	2023-11-14: Updated project metadata.

Dataset with its publication pending

submitter keyword: Glycoproteomics, mucinomics FAIMS, mucin-domain glycoproteomics, enrichment., ion mobility separation

Stacy Alyse Malaker
contact affiliation	Department of Chemistry, Yale University
contact email	stacy.malaker@yale.edu
lab head
Valentina Rangel Angarita
contact affiliation	Graduate Student, Malaker Lab
contact email	valentina.rangelangarita@yale.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD041217
     1. Label: PRIDE project
     2. Name: False positive glycopeptide identification through in-FAIMS glycan fragmentation.