MGL is known to bindterminal residues of N-acetylgalactosamine
To validatethe specificity of the interaction of the proteins listed in Table with MGL , we nextevaluated our data for the presence of glycopeptides carrying thismotif
During our LC–MS/MS analyses of the tryptic digests,we used a method that triggered additional MS/MS acquisitions oncethe MS/MS spectrum showed the presence of the characteristic HexNAcoxonium ion at m/z 204.087 ([C8H14NO5]+)
First of all ,for these additional MS/MS events, we used a higher number of ionsfor fragmentation , resulting in a higher signal-to-noise for the fragmentions
Moreover, to improve the confidence of the glycopeptide identifications,we collected Orbitrap spectra using different HCD collision energiesin addition to an ion trap CID spectrum
Next, we searched forglycopeptides carrying the Tn antigen following database searchesusing MaxQuant and Byonic
With these approaches, we could confirmthe presence of the Tn antigen on CD45 and CD43 (Table ) with peptides containing amaximum of four HexNAcs (Table )
However, it is known that certain regions in these twoproteins contain a high density of O-glycans, which may have beenmissed by the automatic data analysis due to limitations of the particularsoftware used, for example, due to the high number of occupied glycosylationsites in one individual peptide
Indeed, upon manual inspection ofour data, we observed peptides containing up to 11 HexNAcs (Figure )
Of note, one peptidewith an extended O-glycan on the CD45 tryptic peptide LNPTPGSNAISDVPGER(HexNAc2Hex1NeuAc1) was found using Byonic, which, considering the Cosmc mutation in Jurkat cells, could correspond to a core 6 O-glycan,a specific MGL-binder (GlcNAcβ1-6GalNAcαSer/Thr)
In addition to CD43 and CD45 , our database searches confirmedthepresence of the Tn antigen on another 11 proteins (Table )
We confirmed the Tn antigenon all of these peptides by manual interpretation of the data
Asan example, two glycopeptides from EVI2B and TREML2 , respectively,are shown in Figure
Between the peptides presented in Table , one peptide (GLFIPFSVSSVTHK)with three HexNAcs was identified from P-selectin glycoprotein ligand1 ( SELPLG ), which was not identified as a specific binder in the proteomicsdata (Table )
Inspectionof the data showed that, unexpectedly, it was filtered out due tothe fact that normal tryptic peptides of SELPLG were observed in onlyone of the three MGL pull-downs
Terminal N-acetylgalactosamines can alsobe partof N-glycans, for example, as part of the LacdiNAcepitope
Hence we also searched our data for the presence of N-glycopeptides using Byonic
Only for CD45 presented in Table we could identify N-glycopeptides (on N234 , 278, 337, 380, 421, and 470, respectively),but none of these appear to contain a terminal N-acetylgalactosamine ,as judged on the basis of the glycan com positions as well as the absenceof the LacdiNAc (GalNAcβ1-4GlcNAcβ1) marker ion at m/z 407.166 (Table S2)