Stable Isotope Dilution Multidimensional Liquid Chromatography-Tandem Mass Spectrometry for Pancreatic Cancer Serum Biomarker Discovery

Abstract

A novel approach to pancreatic cancer biomarker discovery has been developed, which employs a stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation coupled with tandem mass spectrometry (MS/MS). Secreted proteins from CAPAN-2 human pancreatic cancer derived cells were collected after conducting stable isotope labeling by amino acids in cell culture (SILAC). The resulting SILAP standard contained <0.5% of individual unlabeled proteins. Pooled sera from patients with early stage pancreatic cancer or controls were prepared, and an equal amount of the SILAP standard was added to each sample. Proteins were separated by isoelectric focusing (IEF) prior to two-dimensional liquid chromatography (2D-LC)–MS/MS analysis. A total of 1065 proteins were identified of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer. ELISA validation of these findings was successfully performed for two proteins, ICAM-1 and BCAM. Results of these studies have provided proof of principle that a SILAP standard derived from the CAPAN-2 secreted proteome can be used in combination with extensive multidimensional LC-MS/MS for the identification and relative quantitation of potential biomarkers of pancreatic cancer. This technique allows for the detection of low-abundance proteins, and focuses only on biologically relevant proteins derived from pancreatic cancer cells.

Serum metabolomics as a novel diagnostic approach for pancreatic cancer

“…Thus far, many researchers have attempted to find novel biomarkers for pancreatic cancer using proteomic and transcriptomic approaches (Hibi et al 2009;Sitek et al 2009;Yu et al 2009), but their research findings have not demonstrated a practical use. Based on the findings of our current study, it was indicated that the pathogenesis of pancreatic cancer leads to variations in the serum levels of low molecular weight metabolites, and our study supports the potential of metabolomics as an early diagnostic tool for pancreatic cancer, although larger studies are needed to verify its practical utility.…”

Proteomic profiling of renal allograft rejection in serum using magnetic bead–based sample fractionation and MALDI-TOF MS

“…It remains unclear to date whether this complex peptidome may provide a robust correlation of biological events occurring in the entire organism. Therefore, this study aimed at identifying biomarkers that represent renal pathological status by taking the advantage of MS, which allows us to distinguish hundreds of small-to medium-sized peptides in microliters of serum [12,13].…”

Candidate Serological Biomarkers for Cancer Identified from the Secretomes of 23 Cancer Cell Lines and the Human Protein Atlas

“…However, the progress of these studies has been hampered by the complex nature of serum/plasma samples and the large dynamic range between the concentrations of different proteins (14). As cancer biomarkers are likely to be present in low amounts in blood samples, the direct isolation of these markers from plasma and serum samples requires a labor-intensive process involving the depletion of abundant proteins and extensive protein fractionation prior to mass spectrometric analysis (15)(16)(17)(18). Alternatively, the secretome, or group of proteins secreted by cancer cells (19), can be analyzed to identify circulating molecules present at elevated levels in serum or plasma samples from cancer patients.…”