One-step synthesis of hydrophilic microspheres for highly selective enrichment of N-linked glycopeptides

A polyacrylamide-based hydrophilic microsphere with a lot of hydroxyl groups on surface (PAM-OH HMS) was prepared in one step. The synthetic process was simple reverse suspension polymerization without any chemical derivation or grafting steps. The properties of obtained HMS were characterized by scanning electron microscopy (SEM), static water contact angle measurement, and FT-IR. The abundant hydroxyl groups on the surface make the material highly good hydrophilic and thus it was utilized for N-glycopeptides enrichment. The enrichment efficiency of PAM-OH HMSs was demonstrated by capturing N-linked glycopeptides from tryptic digest of human immunoglobulin G (IgG). The detection sensitivity for N-glycopeptides identification by MALDI-TOF MS was as low as 10 fmol for tryptic digest of standard human IgG. The selectivity of the HMS towards N-glycopeptides had almost no decrease when the molar ratio of BSA tryptic digest to IgG tryptic digest was increased from 10:1 to 100:1. Moreover, in the LC-MS/MS analysis of real biological sample, a total of 344 unique N-glycosites in 598 unique N-glycopeptides from 172 N-glycoproteins were identified from 2 μL human serum after deglycosylated by PNGase F, and 825 intact N-glycopeptides with different types of glycoform were detected when directly analyzed the N-glycopeptides enriched by PAM-OH HMS. To show the potential of our material in solving real biological issues, N-glycopeptides in the serum from hepatocelluar carcinoma (HCC) patient and health control were enriched and quantified. All the experiments demonstrated that this polyacrylamide-based hydrophilic microsphere shows a great potential to enrich the low-abundance N-glycopeptides for glycoproteome analysis of real complicated biological samples.