Sylvie Pons

Identification and verification of heat shock protein 60 as a potential serum marker for colorectal cancer

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two‐dimensional difference gel electrophoresis (2D‐DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione‐S‐transferase Pi, α‐enolase, T‐complex protein 1 subunit β, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5–15%) and sensitive (0.3 ng·mL−1) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver‐operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three‐marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19‐9 reached 0.77. Serum HSP60 appeared to be more specific for late‐stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

Analysis of high molecular mass proteins larger than 150 kDa using cyanogen bromide cleavage and conventional 2‐DE

Proteomic approaches including high‐resolution 2‐DE are providing the tools needed to discover disease‐associated biomarkers in complex biological samples. Although 2‐DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2‐DE in polyacrylamide gels. This strategy is based on the 2‐DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2‐DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with Mr comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2‐DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.