Integrated Glycoproteomics Demonstrates Fucosylated Serum Paraoxonase 1 Alterations in Small Cell Lung Cancer

Abstract

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and the detection of SCLCs at an early stage is necessary for successful therapy and for improving cancer survival rates. Fucosylation is one of the most common glycosylation-based modifications. Increased levels of fucosylation have been reported in a number of pathological conditions, including cancers. In this study, we aimed to identify and validate the aberrant and selective fucosylated glycoproteins in the sera of patients with SCLC. Fucosylated glycoproteins were enriched by the Aleuria aurantia lectin column after serum albumin and IgG depletion. In a narrowed down and comparative data analysis of both label-free proteomics and isobaric peptide-tagging chemistry iTRAQ approaches, the fucosylated glycoproteins were identified as up- or down-regulated in the sera of limited disease and extensive disease stage patients with SCLC. Verification was performed by multiple reaction monitoring-mass spectrometry to select reliable markers. Four fucosylated proteins, APCS, C9, SERPINA4, and PON1, were selected and subsequently validated by hybrid A. aurantia lectin ELISA (HLE) and Western blotting. Compared with Western blotting, the HLE analysis of these four proteins produced more optimal diagnostic values for SCLC. The PON1 protein levels were significantly reduced in the sera of patients with SCLC, whereas the fucosylation levels of PON1 were significantly increased. Fucosylated PON1 exhibited an area under curve of 0.91 for the extensive disease stage by HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The glycan structural analysis of PON1 by MS/MS identified a biantennary fucosylated glycan modification consisting of a core + 2HexNAc + 1Fuc at increased levels in the sera of patients with SCLC. In addition, the PON1 levels were decreased in the sera of the Lewis lung carcinoma lung cancer mouse model that we examined. Our data suggest that fucosylated protein biomarkers, such as PON1, and their fucosylation levels and patterns can serve as diagnostic and prognostic serological markers for SCLC.

Quantitative Profiling of Post-translational Modifications by Immunoaffinity Enrichment and LC-MS/MS in Cancer Serum without Immunodepletion

“…Diagnostics research has focused on identifying biomarkers from viable biofluids, including serum/plasma, saliva, cerebrospinal fluid, and urine. Due to ease of collection and richness in proteins and metabolites, serum/plasma has been the preferred choice for diagnostic studies (1)(2)(3). Advancement of mass-spectrometry-based proteomic technologies has allowed identification and quantification of thousands of proteins in serum/plasma samples.…”

Applications of Multiple Reaction Monitoring to Clinical Glycomics

“…The validation of differentially fucosylated glycoproteins that were previously identified was assessed in a similar fashion [28]. Using AAL lectin, followed by tryptic digestion and MRM analysis of the respective peptides the four proteins APCS, C9, SERPINA4, and PON1 were shown to be differentially fucosylated in sera of non-small cell lung cancer patients [28] as illustrated in Figure 1 for PON1.…”

“…Using AAL lectin, followed by tryptic digestion and MRM analysis of the respective peptides the four proteins APCS, C9, SERPINA4, and PON1 were shown to be differentially fucosylated in sera of non-small cell lung cancer patients [28] as illustrated in Figure 1 for PON1. An alternative strategy for the use of MRM in glycoprotein analysis is based on the observation that differential glycosylation may result in altered levels of missed cleavages [29, 30].…”

“…The P-values were calculated using nonparametric Kruskall-Wallis analysis of variance. Reprinted from [28]. …”

Roles for Paraoxonase but not Ceruloplasmin in Peritoneal Washing Fluid in Differential Diagnosis of Gynecologic Pathologies

“…PON1 activity has been demonstrated to be reduced in patients with coronary artery diseases, hypercholesterolemia, type-2 diabetes and iron deficiency. In clinical studies, a lower serum PON1 activity has been observed in patients with lung, pancreas, gastric and prostate cancer compared to the control groups (Ahn et al, 2013).…”