Glycobiology Advance Access published online on February 13, 2008
Glycobiology, doi:10.1093/glycob/cwn013
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N-Glycoproteomics – an automated workflow approach
1 MediCel, Haartmaninkatu 8, FIN-00290 Helsinki, Finland
2 Transplantation laboratory, Haartman Institute, P.O. Box 63, FIN-00014 University of Helsinki
3 HUCH Laboratory Diagnostics, Helsinki University Central Hospital, P.O. Box 401, FIN-00029 HUCH, Helsinki, Finland
To whom correspondence should be addressed: Haartman Institute, P.O. Box 63, FIN-00014 University of Helsinki, Helsinki. Tel.: (+358) 9 1912 5111; Fax (+358) 9 1912 5155; E-mail risto.renkonen{at}helsinki.fi
Received on November 22, 2007; accepted on February 3, 2008
Glycan decorations dictate protein functions and thus have crucial importance in life sciences. Previously glycoprotein analysis has mainly focused on analysis the liberated glycans allowing detailed structural, but lacking positional information. Analysis of intact glycopeptides has required purified glycoproteins and manual interpretation of spectra. We developed an approach where mixtures of native glycopeptides were analysed with tandem mass spectrometry and the spectra were analysed with automated in silico workflows. The latter included combination of original spectra, generation of a human N-glycopeptide library, matching glycopeptide spectra to the theoretical peptide fragments, scoring the observations, predicting the glycan composition, which were then matched against observed spectra, statistical validation of the results with target-decoy filtering and finally calculation of glycan structures. We verified this approach with 150 serotransferrin glycopeptide spectra, where we automatically generated 105 putative interpretations from > 109 theoretical glycopeptides. After scoring 62 glycopeptide spectra obtained validated interpretation with concomitant amino acid sequences, glycan compositions and structures. When applying this method to an unknown mixture of human plasma glycoproteins we identified 80 glycopeptides with their glycan compositions or structures. Instead of weeks and months of interpretation work of mass spectrometry files our automated workflow can be executed in few hours and provide information concomitantly from both the amino acid and glycan moieties of intact glycopeptides in mixtures. No advanced computational skills were needed to use these preformed and tested workflows. In case users want to add complexity to the analysis they are allowed to alter all parameters and rebuild the workflows.
Key words: N-glycoproteomics / glycopeptide / mass spectrometry / automated workflow