A label-free nano-liquid chromatography–mass spectrometry approach for quantitative serum peptidomics in Crohnʹs disease patients

The identification of serum biomarkers for the diagnosis of inflammatory bowel diseases able to reduce the need for invasive tests represents a major goal in their therapy and follow-up. We report here a methodological approach for the evaluation of specific changes in the serum peptides abundance in healthy (H) and Crohnʹs disease (CD) subjects, based on a label-free LC ESI/Q-TOF differential mass spectrometry (MS) approach combined with targeted MS/MS analysis. The low molecular weight serum proteins were separated by RP nano-LC ESI/Q-TOF MS and the resulting datasets were aligned with msInspect software. The differently abundant peptides, evaluated using Proteios Software Environment, were identified by MS/MS analysis and database search. The identification of clusters of peptides resulting from proteins (such as fibrinogen-α) commonly involved in physiological processes lead to the evaluation of a possible role in CD of specific serum exoproteases. An assay based on synthetic peptides spiked into H, CD and ulcerative colitis (UC) serum samples as substrate, followed by MALDI MS and chemometric analysis of the metabolite patterns has been developed achieving a 100% discrimination between CD, UC and H subjects. The results are promising for the application of this approach as a simple tool for diagnostic aims and biomarker discovery in CD.