Glycoproteins: An Innovative Approach to Analysis

Glycosylation is a posttranslational modification whose analysis has implications in understanding many biochemical processes and both inter- and intracellular interactions. The analysis of glycopeptides also has clinical significance for the therapeutic treatment of medical issues. In particular, expression changes in glycoproteins have been observed in some cancers, allowing researchers to identify and plot the progression of the disease over time and in response to treatment. Some cancer tumors secrete proteins with altered glycans that form the basis for a concerted immune response with the production of antibodies to these abnormal modifications. These antibodies may serve as diagnostic markers.¹ Another clinical implication is the search for an effective vaccine to eradicate communicable diseases, like HIV. Some believe that the virus’ envelope of evolving glycosylated proteins may hold the key to unlocking vaccination protocols.²

The traditional methods for analyzing these profoundly important glycoproteins come with problematic restrictions. This has limited researchers to piecemeal investigations of the peptide backbone and glycosylation sites. For instance, collision-activated dissociation (CAD) cleaves the peptide-glycan bond and eliminates the opportunity for researchers to analyze the intact glycoprotein and the glycosylation site. Utilizing higher energy collision dissociation (HCD) in concert with electron transfer dissociation (ETD) allows researchers to identify glycopeptides, but the multi-step process can be tedious and inefficient. One of the problems with this method is that once researchers obtain HCD spectra, they must interrogate the entire spectra for the oxonium ions that indicate the presence of glycopeptides without exercising selectivity. An additional concern is that isobaric ions may trigger false positives.³

Higher-energy collision dissociation-accurate mass-product dependent electron transfer dissociation (HCD-PD-ETD) represents one approach to the issues inherent in traditional glycopeptide analysis. Research by Saba et al. indicates that HCD-PD-ETD can more efficiently and accurately produce data. Using an LTQ Orbitrap velos hybrid mass spectrometer with ETD (Thermo Scientific), the researchers were able to interrogate the HCD spectra for oxonium ions and intelligently apply ETD spectra only when a glycopeptide precursor was identified. This increases productivity and may even enable the identification of low level glycopeptides. In addition, the high mass accuracy effectively eliminates false positives.

Saba et al. compared the results gathered with HCD-PD-ETD with the results of traditional HCD/ETD protocols in the analysis of a 12-protein mixture containing two glycoproteins and two contaminants. When compared to the traditional method, the researchers found that the conventional approach failed to recognize the glycopeptides from the contaminant glycoproteins. They believe that HCD-PD-ETD allowed them to identify low levels of contaminant glycopeptides because the streamlined nature of the approach eliminated the need to perform ETD on non-glycosylated peptides. The increased productivity paved the way for greater accuracy.

HCD-PD-ETD allows researchers to more accurately and efficiently analyze the structure of glycoproteins. This advancement in proteome research has implications on a wide variety of functional applications, including the therapeutic potential of glycopeptides analysis as a marker for disease and a tool for vaccine research.

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