Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides

J Proteome Res. 2011 May 6;10(5):2248-60. doi: 10.1021/pr101082c. Epub 2011 Mar 28.


Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid / methods
  • Glycopeptides / chemistry*
  • Glycopeptides / metabolism
  • Glycoproteins / chemistry*
  • Glycoproteins / metabolism
  • Glycosylation
  • Humans
  • Peptide Hydrolases / metabolism*
  • Proteomics / methods*
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Tandem Mass Spectrometry / methods


  • Glycopeptides
  • Glycoproteins
  • Peptide Hydrolases