Elucidation of N-Glycosites Within Human Plasma Glycoproteins for Cancer Biomarker Discovery

Abstract

Glycans are an important class of post-translational modifications that decorate a wide array of protein substrates. These cell-type specific molecules, which are modulated during developmental and disease processes, are attractive biomarker candidates as biology regarding altered glycosylation can be used to guide the experimental design. The mass spectrometry (MS)-based workflow described here incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan motifs. The goal was to design a relatively simple method for the rapid analysis of small plasma volumes (e.g., clinical specimens). As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin and AAL, which bind sialic acid- and fucose-containing structures, respectively. Positive controls (fucosylated and sialylated human lactoferrin glycopeptides), and negative controls (high-mannose glycopeptides from Saccharomyces cerevisiae invertase) were used to monitor the specificity of lectin capture and optimize the workflow. Multiple Affinity Removal System 14-depleted, trypsin-digested human plasma from healthy donors served as the target analyte. Samples were loaded onto the lectin columns and separated by high performance liquid chromatography (HPLC) into flow through and bound fractions, which were treated with PNGase F, an amidase that removes N-linked glycans and marks the underlying asparagine glycosite by a +1 Da mass shift. The deglycosylated peptide fractions were interrogated by HPLC ESI-MS/MS on a quadrupole time-of-flight mass spectrometer. The method allowed identification of 122 human plasma glycoproteins containing 247 unique glycosites. Notably, glycoproteins that circulate at ng/mL levels (e.g., cadherin-5 at 0.3-4.9 ng/mL, and neutrophil gelatinase-associated lipocalin which is present at ∼2.5 ng/mL) were routinely observed, suggesting that this method enables the detection of low-abundance cancer-specific glycoproteins.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…Detection of the sites of attachment of the glycans is another area amenable to MALDI and other mass spectrometric methods. Practical details of a method for identification of N ‐glycosites by monitoring the Asn to Asp conversion following PNGase F release of the glycans have been published (Drake et al, ). The method has been used by Goto et al () to show that human hyaluronidase 1 is glycosylated at three sites.…”

Abundance-Ratio-Based Semiquantitative Analysis of Site-Specific N-Linked Glycopeptides Present in the Plasma of Hepatocellular Carcinoma Patients

“…There are two common mass spectrometry (MS)-based approaches for characterization of glycoproteins. One approach involves comprehensive identification of N-linked glycopeptides after the release glycans by PNGase F treatment. − The N-linked site can then be identified on the basis of the difference in mass from the native sequence associated with conversion of Asn to Asp (0.98 Da). However, this indirect approach does not yield information about the glycan structure and therefore does not reveal the potential influence of glycan variability on the disease process.…”

Characterization of the hypersensitive response‐like cell death phenomenon induced by targeting antiviral lectin griffithsin to the secretory pathway

“…Like GRFT, their pharmaceutical activities depend on their mannose binding activities (Ferguson et al ., ; Larsen et al ., ; Liu et al ., ). Several lectins that bind high mannose glycans, including GRFT and CV‐N, accumulate well in various plants such as tobacco, marshmallow, soya bean and rice, and do not cause necrosis symptoms (Drake et al ., ; O'Keefe et al ., , ; Vamvaka et al ., ). These studies support our data that demonstrate that cytosolic‐targeted expression of lectins do not induce necrotic responses in the host plant.…”