Identification of a novel serum biomarker for pancreatic cancer, C4b-binding protein α-chain (C4BPA) by quantitative proteomic analysis using tandem mass tags.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease due to the lack of specific early diagnostic markers. To improve the outcomes, proteomic approaches are being developed for the discovery of novel biomarkers of PDAC. METHODS: Using tandem mass tag labelling and LC-MS/MS, we performed comparative analyses of pre- and postoperative sera from PDAC patients to identify specific serum biomarkers for PDAC. In validation studies, we evaluated the discriminatory power of candidate proteins. RESULTS: Among the 302 proteins analysed, 20 were identified as potential biomarkers, with C4b-binding protein α-chain (C4BPA) and polymeric immunoglobulin receptor (PIGR) being selected for further analysis. The sera levels of C4BPA and PIGR were significantly higher in the preoperative PDAC patients than in the postoperative ones (P<0.008, P<0.036, respectively). In addition, serum C4BPA levels, but not PIGR, in patients with PDAC were significantly higher than those in healthy controls as well as in patients with pancreatitis and other malignancies including biliary tract cancers (BTC) (P<0.001). The respective area under the receiver operator characteristics (ROC) curve (AUC) was 0.860 for C4BPA, 0.846 for CA19-9 and 0.930 for the combination of C4BPA and CA19-9 in PDAC vs non-cancer individuals. The respective AUC was 0.912 for C4BPA, 0.737 for CA19-9 in Stages I and II of PDAC, 0.854 for C4BPA and 0.264 for CA19-9 in PDAC vs BTC. CONCLUSIONS: We have demonstrated that C4BPA is a novel serum biomarker for detecting early stage PDAC, as well as for distinguishing PDAC from other gastroenterological cancers. Further analysis in large cohort studies will warrant C4BPA as a promising biomarker of PDAC in clinical use.

Evaluation of serum carbohydrate-deficient transferrin by HPLC and MALDI-TOF MS.

BACKGROUND: The percentage of carbohydrate-deficient transferrin (%CDT) in serum is a marker of habitual alcohol intake that can be determined by antibody detection of abnormal disialo sugar chains at D432 and D630. However, this approach lacks specificity for alcoholic liver disease. To decrease the false-positive rate in patients with non-alcoholic liver diseases, we developed a screening method using the disialo sugar chain at D630 alone. METHODS: Serum was obtained from 12 patients with alcoholic liver disease, 12 with type C chronic liver disease, 6 with non-alcoholic steatohepatitis (NASH), and 12 healthy non-alcohol drinkers. Transferrin with two sialic acids (disialotransferrin) was fractionated from serum using HPLC, digested with trypsin, and evaluated using MALDI-TOF MS. RESULTS: An abnormal sugar chain at D630 of transferrin was not detected in healthy subjects or in patients with chronic liver disease or NASH, but was detected in 9 patients (75%) with alcoholic liver disease. Positive results were found in 3 samples that were negative using an N-Latex CDT kit and in one sample negative for γ-glutamylaminotransferase and CDT. CONCLUSIONS: Detection of CDT by HPLC/MALDI-TOF MS based on an abnormal sugar chain at D630 may permit identification of habitual alcohol drinkers when used in combination with current markers.

Adsorption of urinary proteins on the conventionally used urine collection tubes: possible effects on urinary proteome analysis and prevention of the adsorption by polymer coating.

One possible factor determining recovery of trace amount of protein biomarker candidates during proteome analyses could be adsorption on urine tubes. This issue, however, has not been well addressed so far. Recently, a new technical device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (poly(MPC-co-BMA)) has been developed mainly to prevent the adsorption of plasma proteins. We assessed whether conventionally used urine tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(MPC-co-BMA) can minimize the adsorption. Proteinuric urine samples were kept in poly(MPC-co-BMA)-coated and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or peptides onto urine tubes were analyzed by SDS-PAGE, 2-DE, and the MALDI-TOF MS. It was found that a number of proteins and/or peptides adsorb on the conventionally used urine tubes and that surface coating by poly(MPC-co-BMA) can minimize the adsorption without any significant effects on routine urinalysis test results. Although it remains to be clarified to what extent the protein adsorption can modify the results of urinary proteome analyses, one has to consider this possible adsorption of urinary proteins when searching for trace amounts of protein biomarkers in urine.