Urine is a complex mixture of metabolites of various chemical nature and thus a significant source of biomarkers for human diseases. Short endogenous peptides are amino acid sequences shorter than four which have been frequently described as potential biomarkers, but still represent an analytical challenge due to their wide range of polarity, poor ionization efficiency using electrospray-mass spectrometry (MS) for their determination and the absence of automation in their detection. The aim of the present project will be the development of an analytical methodology for the enrichment, separation and identiffication of short peptides from urine samples in order to tackle the main issues related to the analysis of these compounds. First, an enrichment strategy based on graphitized carbon black tested and developed on a mixture of analytical standard peptides which was representative of the naturally occurring peptides in body fluids. Ultra high performance liquid chromatography separation was carried out using two orthogonal chromatographic strategies, namely reversed phase (RP) C18 and Zic-HILIC columns, in order to maximize the number of identified peptides. A suspect screening approach was chosen for high resolution MS coupling.[3] In particular, a database of all the amino acid combinations for short peptides was compiled and MS/MS fragmentation was only performed on precursor ions matching with those in the database, resulting in a significant boost in sensitivity. Finally, MS/MS spectra were manually matched to spectra generated in silico to confirm the identity and the correct amino acid sequence. The method will be applied to the investigation of short endogenous peptides in human urine from healthy individuals. After the optimization of the methodology,for assessing a specific pathology, eligible participants, including pathological patients will be collected to discover possible new biomarkers related to a specific pathology.
