Short peptides are of extreme interest in clinical and food research fields; nevertheless, they still represent a crucial analytical issue. This lecture aims to provide an overview of analytical solutions developed for enrichment, separation, and untargeted identification of short peptides in biological matrices (i.e.urine) and food matrices. Direct analysis of short peptides is not usually feasible due to the potential association with high abundant components, such as proteins, and the low abundance of peptides compared to other molecules, which can cause extensive ion suppression during electrospray ionization (ESI). A dedicated protocol for enrichment and clean-up of short peptides can overcome some of these issues. In particular, the use of carbonaceous material for the enrichment and separation will be discussed, highlighting the advantages of retaining polar, non-polar, acidic, and positively zwitterionic charged compounds due to strong hydrophobic and ion-exchange interaction . However, sample clean-up is not straightforward either due to the heterogeneous nature of short peptides in polarity, hindering their pre-concentration from complex matrices and chromatographic separation. Alternative separation strategies will be considered to improve the coverage of short polar peptides. Another aspect that will be discussed in detail will be developing a database implemented in Compound Discoverer 3.0, a software dedicated to the analysis of short molecules, to create a data processing workflow specifically dedicated to short peptide tentative identification. Finally, an untargeted metabolomics approach of prostate cancer zwitterionic and positively charged compounds in urine will be presented .
The work was supported by the PRIN project Prot. 2017Y2PAB8, entitled “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”, provided by the Italian Ministry of Education, Universities and Research.
 S. Piovesana, A.L. Capriotti, et al. Anal Chem, 45, (2019), 11474–11481  A.Cerrato, A.L. Capriotti et al. ACA, 1158 (2021), 338381
Department of Chemistry, University of Roma La Sapienza, Piazzale Aldo Moro,5 00185 Rome, Italy
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