<b>Annotated glycopeptide spectra for "Glycoproteomic and Proteomic analysis of </b><b><i>Burkholderia </i></b><b><i>cenocepacia</i></b><b> reveals glycosylation events within FliF and MotB are dispensable for motility"</b>

<b>Abstract:</b>Across the Burkholderia genus <i>O</i>-linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in <i>Burkholderia cepacia</i> complex species, such as <i>Burkholderia cenocepacia,</i> little is known about how specific glycosylation sites impact protein functionality. Within this study we sought to improve our understanding of the breadth, dynamics and requirement for glycosylation across the <i>B. cenocepacia O-</i>glycoproteome. Assessing the <i>B. cenocepacia</i> glycoproteome across different culture media using complementary glycoproteomic approaches we increase the known glycoproteome to 141 glycoproteins. Leveraging this repertoire of glycoproteins, we quantitively assessed the glycoproteome of <i>B. cenocepacia</i> using Data-Independent Acquisition (DIA) revealing the <i>B. cenocepacia</i> glycoproteome is largely stable across conditions with most glycoproteins constitutively expressed. Examination of how the absence of glycosylation impacts the glycoproteome reveals that the protein abundance of only five glycoproteins (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) are altered by the loss of glycosylation. Assessing Δ<i>fliF</i> (ΔBCAL0525), Δ<i>motB</i> (ΔBCAL0127) and ΔBCAM0505 strains we demonstrate the loss of FliF, and to a lesser extent MotB, mirror the proteomic effects observed in the absence of glycosylation in Δ<i>pglL</i>. While both MotB and FliF are essential for motility we find loss of glycosylation sites in MotB or FliF does not impact motility supporting these sites are dispensable for function. Combined this work broadens our understanding of the <i>B. cenocepacia</i> glycoproteome supporting the loss of glycoproteins in the absence of glycosylation is not an indicator of the requirement for glycosylation for protein function.<b>Supplementary Data 1. Annotated spectra of unique glycopeptides of </b><b><i>B. cenocepacia </i></b><b>K56-2</b><b><i> </i></b><b>identified across</b><b><i> </i></b><b>growth conditions using FAIMS and ZIC-HILIC enrichments. </b>For the best scoring unique glycopeptides identified using MSfragger the data files/spectrum associated with the assigned spectra, peptide length, charge state, observed m/z, delta mass (glycan assignment), MSfragger Hyperscore, assigned modification, Uniprot Protein ID, assigned J2315 gene number, if the EThcD spectra enabled localisation of the glycan within the sequence and the pages of the HCD / EThcD corresponding to the unique glycopeptide sequence provided.<br><b>Supplementary Data 2. Annotated spectra of novel glycopeptides identified within </b><b><i>B. cenocepacia </i></b><b>J2315</b><b><i> </i></b><b>from previous studies</b><b>. </b>For the best scoring unique glycopeptides identified within previous studies yet not manually validated the Uniprot Protein ID, Byonic associated assignment information (m/z; mass error, score, delta score, delta mod score, scan number within the LC-MS run and scan time) in addition to the peptide sequence, assigned J2315 gene number, if the spectra enable localisation of the glycan within the sequence and the pages of the associated HCD / EThcD spectra corresponding to the unique glycopeptide sequence provided.