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.
Highlights
hybrid A. aurantia lectin ELISA (HLE), whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot
Analysis of the Fucosylation Levels of Target Proteins in the Sera of Small cell lung cancer (SCLC) Patients—We investigated the levels of the fucosylated forms of the APCS, C9, SERPINA4, and PON1 proteins in the SCLC serum samples
In this study, fucosylated glycoproteins enriched by the AAL column were analyzed by two types of comparative proteomic approaches involving label-free and iTRAQ labeling
Summary
HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The abbreviations used are: SCLC, small cell lung cancer; HE, healthy control; LD, limited disease stage; ED, extensive disease stage; AFP L3 fraction, ␣-fetoprotein L3 fraction; AAL, Aleuria aurantia lectin; TIC, total ion current; AUC, area under curve; LLC, Lewis lung carcinoma; iTRAQ, isobaric tags for relative and absolute quantitation; IPI, International Protein Index; MRM, multiple reaction monitoring; APCS, serum amyloid p component; C9, complement component 9; PON1, serum paraoxonase 1; SERPINA4, kallistatin; HLE, hybrid AAL ELISA; HRP, horseradish peroxidase; PNGase, peptide N-glycosidase F; WB, Western blot; FDR, false discovery rate; Hex, hexose; HexNAc, N-acetylhexosamine; ACN, acetonitrile; ROC, receiver-operator characteristic curve; CID, collision-induced dissociation. 50% of human serum proteins, including secretory and membranebound proteins, are suggested to exhibit various N-linked glycosylation patterns [7] Such glycan structures of proteins during carcinogenesis can affect various aspects of the biological behaviors of tumor cells (8 –11). The sensitive detection of such disease-associated glycosylation changes and abnormalities can become a unique landmark to develop diagnostic and prognostic glycoprotein biomarkers
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