Highly efficient enrichment method for human plasma glycoproteome analyses using tandem hydrophilic interaction...

Selective enrichment of glycopeptides from complex sample with hydrophilic interaction liquid chromatography (HILIC) method, followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites is used extensively as a sample preparation for comprehensive glycoproteome analysis. However, the coenrichment of hydrophilic nonglycosylated peptides and the released N-glycans seriously affect the identification of deglycopeptides with nano-LC-MS/MS. Here, we developed a new method for highly efficient and specific enrichment of human plasma N-glycopeptides using HILIC-PNGaseF-HILIC workflow (HPH). The first HILIC enriches the N-glycopeptides from the complex peptide mixtures. After the enriched N-glycopeptides are deglycosylated with PNGase F, the second HILIC captures the coenrichment of hydrophilic nonglycosylated peptides and the N-glycans, and then further enriches the deglycosylated peptides. The glycopeptide enrichment efficiency can be notably improved by employing HPH, evaluated by the highly recovery (more than 93.6%) and specific capturing glycopeptides from tryptic digest of IgG and BSA up to the molar ratios of 1:200. Meanwhile, we found that the alkylated proteins with IAA can affect the enrichment efficiency for N-glycopeptides with HILIC method. Moreover, after optimism the protein digestion, this novel HPH strategy allowed for the identified 722 N-glycopeptides within 202 unique glycoproteins from 1 µL human plasma digest using PNGase F in H₂¹⁶O. Meanwhile, this new HPH strategy identified an average 501 N-glycopeptides within averagely 134 unique glycoproteins from 1 µL human plasma digest using PNGase F in H₂¹⁸O. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides in the second HILIC. It was found that 52.2% more N-glycosylation peptides were identified by the HPH strategy compared with the using one HILIC enrichment alone.