Mapping the low abundant plasma glycoproteome using Ranachrome-5 immobilized magnetic terpolymer as improved HILIC sorbent

HILIC (hydrophilic interaction liquid chromatography) materials enrich glycopeptides. The non-specific interactions because of support material and inadequate hydrophilicity render loss of less abundant glycopeptides in SPE-based enrichments. In this work, magnetic terpolymer (Fe O @MAA/DVB/1,2-Epoxy-5-hexene) is functionalized with Ranachrome-5 to generate enhanced hydrophilicity. Amine, carboxylic, and amide groups of ranachrome-5 provide zwitterionic chemistry. Material's magnetic core contributes to ease of operation while higher surface area 97.0711 m g immobilizes better quantities of Ranachrome-5. Homogeneous morphology, nano-size, and super hydrophilicity enhance enrichment. Ranachrome-5 functionalized polymeric core-shell beads enrich 25, 18 and 16 N-linked glycopeptides via SPE strategy from tryptic digests of model glycoproteins i.e., immunoglobulin G (IgG), horseradish peroxidase (HRP) and chicken avidin, respectively. Zwitterionic chemistry of ranachrome-5 helps in achieving higher selectivity (1:250, HRP / Bovine Serum Albumin), and lower detection limit (100 attomole, HRP digest) with complete glycosylation profile of each standard digest. High binding capacity (137.1 mg/g) and reuse of affinity material up to seven cycles reduce the cost and amount of affinity material for complex sample analysis. A recovery of 91.76% and relative standard deviation (RSD) values less than 1 define the application of HILIC beads for complex samples like plasma. 508 N-linked intact low abundant glycopeptides corresponding to 50 glycoproteins are identified from depleted human plasma samples via nano-Liquid Chromatography-Tandem Mass Spectrometry (nLC-MS/MS). Using Single Nucleotide Variances (BioMuta) for low abundant plasma glycoproteins, the potential association of proteins to four cancers, i.e., breast, lung, uterine, and melanoma is evaluated. Via the bottom-up approach, HILIC beads can analyze clinically important low-abundant glycoproteins.