Glycopeptides, formed by the addition of glycan groups, are a common post-translational modification (PTM). They are closely associated with various cell activities including cell-to-cell interaction, apoptosis and receptor-ligand binding. Glycan groups are either O-linked through serine or threonine residues or, more commonly, N-linked via asparagine or arginine. Studies have shown that this PTM is often abnormally expressed in disease states such as cancer. In the search for novel biomarkers of disease, it is therefore clinically relevant to examine glycoprotein isoforms for changes in glycosylation site or altered abundance.
Unfortunately, detection and characterization of glycoproteins are not straightforward. They are usually present at low levels in complex samples such as plasma or serum, where matrix and more abundant proteins can mask their presence. Additional complications to mass spectrometric (MS) detection include dilution of precursor ion signal and the lack of a common PTM mass shift.
A Thermo Fisher Scientific research team (Peterman et al., 2014) that included scientists from Eastern Virginia Medical School recently developed an automated workflow using multiplexed liquid chromatography–mass spectrometry (LC-MS) to characterize and quantify O-linked glycopeptides in pooled plasma. They used serial antibody extraction to immune-enrich the analytes, followed by acquisition of high-resolution/accurate mass (HR/AM) and tandem mass spectrometric (MS/MS) data from a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific).
Peterman and co-authors investigated sample preparation methods, comparing straightforward trypsin digestion of unextracted samples with a pre-digestion multiplexed analyte extraction step using mass spectrometric immunoassay immunoprecipitation, or MSIA-IP. They prepared MSIA D.A.R.T.’S pipette tips (Thermo Scientific), each covalently loaded with one of five different antibodies to specific glycopeptides—apolipoprotein CIII, serotransferrin, Zinc-α-2-glycoprotein, α-1-antitrypsin, and lactotransferrin. The scientists compared single extractions with serial extractions; in the former, one MSIA tip was used per plasma well, and in the latter, each well was serially extracted using all five MSIA tips. They then digested the extractions, either pooled together or singly.
Using a targeted data extraction approach implementing automated data reduction, Peterman et al. created a refined list of highly confident peptides from the initial HR/AM and MS/MS results obtained. The scientists used Thermo Scientific’s Proteome Discoverer 1.4 software to identify proteins and peptides and Pinpoint 1.4 software to select O- and N-linked glycopeptides. As an internal quality control, they spiked the samples using the Pierce Peptide Retention Time Calibration (PRTC) kit (Thermo Scientific).
The scientists found that MSIA-IP extraction prior to digestion greatly aided glycopeptide discovery; for example, the area under the curve (AUC) values for the apolipoprotein CIII peptide exhibited a 500-fold increase when compared with the results from the unextracted digests. Furthermore, serial extractions showed similar recovery to single extractions, with the additional benefits of improved workflow and conservation of sample volume.
The scientists propose that the data collected from their new workflow accurately quantifies unmodified peptides and their glycopeptide forms. In addition to conserving limited sample volumes, multiplexed LC-MS shows potential for comparing control samples with those from patients with disease.
1. Peterman, S., et al. (2014) “Targeted Multiplexed Quantitation of Glycoproteins and Corresponding Glycoforms Using MSIA LC-HR/AM MS Analysis,” poster.
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