Enrichment of O-glycoproteins from Human Serum using GlycOCATCH™

Application

Selective enrichment of O-glycoproteins from human serum for improved LC-MS/MS detection and glycosylation-focused proteomic analysis.

Isolation of O-glycoproteins from human serum enables targeted access to a subset of glycosylated proteins that play key roles in cell signalling, immune function, and disease processes. Enriching these species from the complex serum background improves detection sensitivity and supports more detailed characterisation of O-glycosylation patterns in health and disease.

Human serum was treated with SialEXO and incubated with the GlycOCATCH resin. The resin was washed, and O-glycosylated proteins were eluted with 8 M urea. The eluted proteins were denatured, reduced, alkylated and digested with trypsin. Peptides were analyzed using RP-LC-MS/MS and searched against the Swiss Prot database.

 Direct enrichment of O-glycoproteins from human serum

Here we demonstrate the highly efficient enrichment of O-glycosylated proteins which comprise the most abundant proteins identified following the database search. The strong binding affinity of GlycOCATCH demonstrates and effective enrichment strategy for O-glycosylated proteins from the complex human serum matrix. Enrichment of these proteins using GlycOCATCH allows them to be analysed in an improved way using mass spectrometry as these proteins would not necessarily have been so abundantly observed in non-enriched samples, making glycomic analysis much more difficult.

Specific enrichment from complex samples prior to mass spectrometry characterization

Enrichment of O-glycoproteins from human serum. Human serum (Sigma) was treated with SialEXO sialidases and incubated with the GlycOCATCH resin. The eluted proteins were denatured, reduced, alkylated and trypsin digested. Peptides were analyzed using RP-LC-MS/MS on a C18 column (Advance BioPeptide Plus 2.1 x 150mm 2.7 µm from Agilent Technologies) in a 0.1% FA in MQ: 0.1% FA in 95% ACN gradient at 45 °C and a flow of 0.2 ml/min. Detection was on an ESI-Q-TOF Bruker Impact II instrument. Data was converted to mgf format files in Data Analysis Software v 4.4 and searched against the Swiss Prot database using BioPharma Compass v 2.0.1. Identified O-glycosylated proteins and other proteins with >12 matching peptides and a MASCOT score >200 are listed.

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