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 mu L human plasma digest using PNGase F in (H2O)-O-16. Meanwhile, this new HPH strategy identified an average 501 N-glycopeptides within averagely 134 unique glycoproteins from 1 mu L human plasma digest using PNGase F in (H2O)-O-18. 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. (C) 2019 Elsevier B.V. All rights reserved.