Lectin Capture Strategies Combined with Mass Spectrometry for the Discovery of Serum Glycoprotein Biomarkers

Abstract

The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.

Serological AFP/Golgi protein 73 could be a new diagnostic parameter of hepatic diseases

“…29 Therefore maximal GP73 expressions are seen in fully established cirrhosis. 7,8 GP73 mRNA and protein following furin-mediated proteolytic cleavage of N-terminus, is secreted into the serum which are regarded as candidate tumor markers for HCC as reported, 12,14,15,30 But we can observe that no matter what method to use (western blot, ELISA, or RT-PCR), sGP73 raised gradually from healthy controls, hepatitis to LC and HCC.…”

“…Figure 4c shows that sGP73 rose nearly two-fold from the healthy condition (median 48.4 lg/L) to hepatitis (median 73.65 lg/L), gradually to the maximum of 153.8 lg/L (in Child C of LC), indicating continuous liver injury contributes to the elevation of sGP73, in accordance with results on tissue and serum levels. 12,[14][15][16]30 Stimulation of various etiologies, liver parenchymal cells regeneration, and quiescent stellate cells activation and proliferation followed the increase on subcellular Golgi apparatus involved in protein processing and classifying 27 ; meanwhile injury induced an upregulation of endoprotease, 31 and large amounts of GP73 were secreted into blood following furin-mediated proteolytic cleavage of its N-terminus, 32 in parallel with the fibrosis stage. The difference of our result when compared to others was a higher sGP73 levels in LC than in HCC group, maybe causing by the differences of the degree of fibrosis or inflammation of the subjects among various studies.…”

“…GP73 has been considered a useful indicator in evaluating liver impairment or fibrosis. 11 The circulating forms of GP73 (protein and mRNA) give a more sensitive indication than AFP in detecting liver cancer consulting to previous data; [12][13][14][15] nevertheless, sGP73 upregulated gradually from normal to hepatitis and LC groups according to pathologic processes. However, sGP73 in the above investigations was tested by semiquantitative methods, such as western blot.…”

Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification

“…Recent advances in glycoproteomics produced a number of glycoproteins, which have been nominated as disease-related biomarker candidates (14,(43)(44)(45)(46)(47). As a standard procedure employed in concurrent proteomics-based biomarker development, the candidates are first quantified with the samples used in the candidate identification phase, then verified via a hundred clinical samples before the last validation phase (10).…”

Beyond the Sequon: Sites of N-Glycosylation

“…A key mechanism mediating the functional roles of glycans in many biological systems is recognition of specific glycan structures by proteins, or lectins. The specificity of such lectins for defined glycan structures can be used to enrich particular subsets of glycopeptides or glycoproteins bearing those structures (Drake et al, 2006, Zielinska et al, 2010.…”