1. A clean-up strategy for identification of circulating endogenous short peptides in human plasma by zwitterionic hydrophilic liquid chromatography and untargeted peptidomics identification.
- Author
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Piovesana, Susy, Cerrato, Andrea, Antonelli, Michela, Benedetti, Barbara, Capriotti, Anna Laura, Cavaliere, Chiara, Montone, Carmela Maria, and Laganà, Aldo
- Subjects
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HYDROPHILIC interaction liquid chromatography , *LIQUID chromatography , *PEPTIDES , *AMINO acid sequence , *CARBON-black , *CHEMICAL purification , *MASS spectrometry , *SOLID phase extraction - Abstract
• Development of an efficient clean-up strategy for short peptides in plasma. • Comparison of 4 different clean-up protocols (advantages and disadvantages). • Untargeted peptidomic approach for short peptide identification in plasma. • Identification of 91 short peptides, the largest number never identified before. • Development of a rapid and simple method for clinical applications. Short peptides, namely di- tri- and tetra peptides, have been proven to play an important diagnostic role in several diseases. Therefore, the development of an analytical approach for their detection and identification is nowadays an important research goal. This paper describes an analytical procedure able to overcome the issues of short peptide isolation, clean-up and identification in plasma samples. Four different protocols were compared and tested to maximize both recovery and total number of identifications of short circulating plasma endogenous peptides. The purified peptides, coming from the four different tested protocols, were separated by zwitterionic hydrophilic liquid chromatography coupled to high-resolution mass spectrometry with the purpose of accomplishing an untargeted investigation based on suspect screening for short peptides in plasma. In particular, the use of Phree™ Phospholipid removal cartridge in combination with a purification step by solid phase extraction on a graphitized carbon black sorbent allowed the identification of the largest number of amino acid sequences (91 short peptides). The clean-up procedure allowed to tackle the issue of the low abundance of such peptides and their suppression during mass-spectrometric analysis. The results indicated that sample preparation is therefore fundamental for short peptide analysis in plasma samples. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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