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Updated: Jul 19, 2025
Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery
Published on: April 17, 2012
Mapping the low abundant plasma glycoproteome using Ranachrome-5 immobilized magnetic terpolymer as improved HILIC
Muhammad Salman Sajid1, Shafaq Saleem1, Fahmida Jabeen2
1Department of Oncology, Genomics and Epigenomics Shared Resource, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Abstract:
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 (Fe3O4@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 m2 g-1 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.
