Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a unique group of neurodegenerative diseases of the central nervous system, which include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer, and Creutzfeldt–Jacob disease (CJD) in humans. TSEs are caused by the conversion of a normal cellular prion protein designated PrP^c into an abnormal form PrP^Sc (1,2) and are characterized by spongiform degeneration, reactive astrocytosis and prion protein accumulation in the central nervous system. Although the species barrier provides significant protection from the interspecies transmission of prion disease, the BSE epidemic and the resulting rise in variant CJD (vCJD) illustrates the potential impact of prion disease upon human and economic health. Current validated diagnostics for prion diseases are all postmortem. The "gold standard" in prion diagnosis is immunohistochemistry utilizing antiprion protein antibodies on the obex region of the brain (3). Despite the good specificity and sensitivity of these tests, animals infected with prion disease only can be diagnosed late in the preclinical period when sufficient abnormal PrP^Sc has accumulated in brain tissue.

In recent years, there has been a growing interest in applying mass spectrometry (MS)-based proteomics technology to research on biomarker discovery and clinical diagnostics of diseases such as cancers and neurodegenerative disorders. Body fluids such as plasma and cerebrospinal fluid are rich sources of biomolecules. Investigation of these proteomes can reveal correlations between altered protein expression and certain disorders. The glycoproteome is one of the major subproteomes of blood, where glycoproteins secreted into the bloodstream comprise a major part of the plasma proteome. Functionally, the oligosaccharide moieties of various glycoproteins act as selectivity determinants, playing a fundamental role in many biological processes such as immune response and cellular regulation because cell-to-cell interactions involve sugar–sugar or sugar–protein specific recognition (4). Consequently, aberrant glycosylation has now been implicated in many diseases, including hereditary disorders, immune deficiencies, neurodegenerative diseases, cardiovascular disease, and cancer (5).

Here we present a method that enables isolation, identification, and relative quantification of glycoproteins in blood samples using lectin affinity enrichment, isotopic labeling, and tandem MS. By applying this method, 280 glycoproteins were identified from mouse plasma, among which 49 showed greater than twofold changes in the infected animals compared with the controls. Many of the identified proteins are known to be present at very low abundance, which demonstrates the ability of this method to delve into the plasma proteome.

Sample preparation: C57/Bl6 mice were inoculated intraperitoneally with 50 μL of 10% brain homogenate from infected or control mice. At 108 days post-inoculation, animals were anesthetized with isoflorane and blood was collected by cardiac puncture into EDTA-treated vacutainer tubes. The whole blood was centrifuged at 1000xg for 5 min. Plasma was decanted and immediately frozen in liquid nitrogen. Samples from seven infected and seven control mice were pooled separately.

Materials: Tris hydrochloride, chicken ovalbumin, N-acetyl-D-glucosamine, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, manganese chloride tetrahydrate, formaldehyde, deuterated formaldehyde, and sodium cyanoborohydride were purchased from Sigma-Aldrich (St. Louis, Missouri). Sodium chloride, calcium chloride, sodium acetate, and urea were obtained from Fisher Scientific (Pittsburgh, Pennsylvania). Agarose-bound concanavalin A (Con A, 6 mg lectin/mL gel) and wheat germ agglutinin (WGA, 7 mg lectin/mL gel) were purchased from Vector Laboratories (Burlingame, California). Dithiothreitol (DTT) and sequencing grade modified trypsin were purchased from Promega (Madison, Wisconsin). Iodoacetamide was obtained from MP Biomedicals (Solon, Ohio).