55.
Glycan decorations dictate protein functions and thus have crucialimportance in life sciences. Previously glycoprotein analysiswas mainly focused on the analysis of the liberated glycansallowing detailed structural, but lacking positional information.Analysis of intact glycopeptides required purified glycoproteinsand manual interpretation of spectra. We developed an approachwhere mixtures of native glycopeptides were analyzed with tandemmass spectrometry and the spectra were analyzed with automated
in silico workflows. The latter included combination of theoriginal spectra, generation of a human
N-glycopeptide library,matching the glycopeptide spectra to the theoretical peptidefragments, scoring the observations, predicting the glycan composition,which were then matched against the observed spectra, statisticalvalidation of the results with target–decoy filtering,and finally the calculation of glycan structures. We verifiedthis approach with the 150 serotransferrin glycopeptide spectra,where we automatically generated 10
5 putative interpretationsfrom >10
9 theoretical glycopeptides. After scoring 62 glycopeptidespectra obtained validated interpretation with concomitant aminoacid sequences, glycan compositions, and structures. When applyingthis method to an unknown mixture of human plasma glycoproteinswe identified 80 glycopeptides with their glycan compositionsor structures. Instead of weeks and months of interpretationwork of mass spectrometry files our automated workflow can beexecuted in few hours and provide information concomitantlyfrom both the amino acid and glycan moieties of intact glycopeptidesin mixtures. No advanced computational skills were needed touse these preformed and tested workflows. In case users wantto add complexity to the analysis they are allowed to alterall parameters and rebuild the workflows.
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