Selective glycopeptide profiling by acetone enrichment and LC/MS

• We established a simple and selective method for glycopeptide enrichment.
• This technique is based on the low solubility of glycopeptides in acetone.
• All of the N-glycopeptides were enriched, regardless of their composition.
• Our method is capable of isolating glycopeptides from complex protein mixtures.

LC/MS is commonly used for site-specific glycosylation analysis of glycoproteins in cells and tissues. A limitation of this technique is the difficulty in acquiring reliable mass spectra for glycopeptides, mainly due to their high heterogeneity and poor hydrophobicity. Here, we establish a versatile method for efficient glycopeptide enrichment to acquire reliable mass spectra. Several lines of evidence using model glycoproteins suggest that our method is based on the different solubility between non-glycosylated and glycosylated peptides in acetone. We also provide data showing that the acetone-precipitated glycopeptide enrichment was successful in acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide in crude human serum. We propose that this method is a powerful tool for the acquisition of reliable mass spectra from trace amounts of glycopeptides and an alternative to lectin affinity enrichment.Biological significanceIn this study, we established a versatile method for glycopeptide enrichment to acquire reliable mass spectra because of the limitation of conventional enrichment methods, such as lectin affinity chromatography. Our enrichment method is capable of isolating glycopeptides from complex peptide mixtures such as crude serum.

Figure optionsDownload high-quality image (171 K)Download as PowerPoint slide