A clean-up strategy for identification of circulating endogenous short peptides in human plasma by zwitterionic hydrophilic liquid chromatography and untargeted peptidomics identification
Introduction
Nowadays, peptidomic analysis has become an independent approach in the omics science in several research fields, such as therapeutics [1] diagnostics [2] and nutraceuticals [3] especially for the biological significance of peptides. Small peptides, namely those possessing 2–4 amino acids in their sequence, have been specifically addressed in food and related for the discovery of new bioactive peptides [4], [5], [6], [7], [8]. Biofluids have currently been considerably less investigated, nevertheless diagnostics has been emerging as a considerable research field due to the role that circulating peptides can play in different diseases, such as pancreatic neuroendocrine tumor [9], papillary thyroid cancer [10], acute leukemia [11], amyotrophic lateral sclerosis [12] and Alzheimer's disease [13,14]. The identification of peptides is therefore important because they can mediate specific functions and enable peripheral detection and they play a crucial role in the progression and spread of the disease, being involved in the processes of cancer cell growth, angiogenesis, and metastasis [15]. Finally, amino acid sequences can be considered biomarkers for several diseases also in personalized medicine approaches [16].
Nevertheless, all peptidomics studies were focused on medium size peptide analysis, since detection and identification of short peptides is still an analytical challenge due to the wide range of polarity of these peptides and their low concentration in complex biological samples [17]. It is well known that small peptides, are nowadays of great importance because of their contribution in various biological processes, including protein synthesis, fertility, neurotransmission, inflammation process, pathogenic microorganism activities and other functions. These functions have made these peptides vital molecules in drug development and health care. Besides, these peptides are also being used as biological markers in biological systems [18].
Despite this, only metabolomics studies indicated some short peptides, together with other molecules, as possible biomarkers [18,19]; only three recent peptidomics papers were specifically meant for 2–4 amino acid long native peptides, one in plasma for diagnosis and prognosis of epithelial ovarian cancer [20], one in serum, for the development of an analytical platform for short peptides detection [21], and one in urine, for the selective enrichment of very short peptides [22]. As far as plasma or serum was concerned, in both papers a simple protein precipitation followed by chromatographic separation (either by C18 revesed phase (RP) or on porous graphitic carbon) was carried out, leading to the identification of a low number of peptides, 15 and 32, respectively. The low number of identifications was probably a consequence of the low electrospray ionization (ESI) efficiency, low concentration and quick degradation of such peptides in the peripheral blood. In fact, a loss of information can occur due to the suppression of short peptides caused by high abundant species, such as proteins and lipids. To overcome those issues, a pretreatment of the sample is mandatory and several strategies have been proposed for the selective removal of high abundance proteins and enrichment of low abundance ones [23].
There are some analytical issues related to the analysis of short amino acidic sequences: their wide range of polarity and concentration make their detection difficult if dedicated protocols for enrichment and clean-up are not performed. Moreover, the bioinformatic identification is not possible with the common databases employed in metabolomics or proteomics, since metabolomics databases currently do not cover all the 168,400 possible combinations of proteinogenic amino acids in di-, tri- and tetrapeptides and proteomic software furnish a too low identification score for short peptide sequences [8,24].
In this context, an analytical platform for the extraction and clean-up of circulating endogenous short peptides in plasma was developed. Four different analytical clean-up methodologies was tested and compared in order to maximize the results in terms of small peptide identification. The best results were achieved by the means of simultaneous protein precipitation and phospholipid removal using the commercial Phree™ Phospholipid removal cartridge, followed by a clean-up step of short proteinogenic peptides by graphitized carbon black (GCB) solid phase extraction (SPE). The chromatographic separation of both hydrophilic and hydrophobic short peptides was carried out by ultra-high performance liquid chromatography (UHPLC) by zwitterionic hydrophilic interaction chromatography (HILIC) coupled tohigh resolution MS (HRMS).Short peptide identification was carried out by an untargeted investigation based on suspect screening. More specifically, a database with all combinations of the 20 natural amino acids within di-, tri- and tetrapeptides was compiled and fragment ions spectra were matched to in silico fragmentation to identify the precursor ions.
Section snippets
Chemicals and reagents
Optima® LC-MS grade water, acetonitrile (ACN) and methanol (MeOH) were supplied from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Trifluoroacetic acid (TFA) was purchased by Romil Ltd (Cambridge). Formic acid (FA), ammonium formate and human plasma were supplied by Sigma-Aldrich (Germany). Dichloromethane (DCM) was provided by VWR International (Milan, Italy). Cartridges packed with 500 mg Carbograph 4 were supplied from Lara S.R.L (Lara S.r.l., Formello, RM, Italy). Phree™
Untargeted peptidomic workflow
Few studies investigated the identity of short peptides by means of HRMS untargeted approaches. In particular, two untargeted peptidomics approaches have been carried out in our research lab by UHPLCHRMS for the analysis of serum and milk short endogenous peptides [21,27], and one work focused on short peptides in plasma as possible diagnostic and prognostic biomarkers in ovarian cancer [20]. Apart from the cited works, the majority of plasma peptidomic studies are, instead, based on database
Conclusions
In this study we investigated the endogenous circulating short peptides in human plasma samples by an untargeted peptidomics approach based on UHPLCHRMS. This study demonstrated that exquisite attention needs to be paid to the methodology selection for the preparation of plasma samples, because the determination of low-abundant compounds, such as short peptides, greatly benefits from the removal of endogenous interfering compounds, such as lipids and salts. In this regard, the wide range of
CRediT authorship contribution statement
Susy Piovesana: Writing - original draft. Andrea Cerrato: Data curation, Software. Michela Antonelli: Methodology. Barbara Benedetti: Methodology. Anna Laura Capriotti: Conceptualization, Funding acquisition. Chiara Cavaliere: Writing - review & editing. Carmela Maria Montone: Methodology. Aldo Laganà: Conceptualization, Funding acquisition.
Declaration of Competing Interest
We have no conflict of interest to declare.
Acknowledgements
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.
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2023, TrAC - Trends in Analytical ChemistryPeptidomics as a tool to analyze endogenous peptides in milk and milk-related peptides
2022, Food BioscienceCitation Excerpt :Regarding the enrichment of endogenous short peptides, due to their low concentration in the matrix and wide range of polar distribution (Capriotti et al., 2016; Piovesana et al., 2020), there are some difficulties in both enrichment and chromatography-mass spectrometry analysis of peptide samples. At present, solid phase matrices of endogenous short peptides have been studied, such as cotton wool, graphitized carbon black, and PhreeTM phospholipid cartridges (Montone et al., 2019; Piovesana et al., 2020). Piovesana and coworkers compared 4 clean-up strategies to extract the short peptides, which were ACN precipitated protein, GCB solid phase extraction, using a PhreeTM Phospholipid cartridge to remove proteins and phospholipids, and commercial PhreeTM Phospholipid combined with GCB solid phase extraction.
Recent developments in peptidomics for the quali-quantitative analysis of food-derived peptides in human body fluids and tissues
2022, Trends in Food Science and TechnologyCitation Excerpt :To improve separation of hydrophilic peptides without pre-column derivatization, dedicated C18-functionalized silica-based resins bearing hydrophobic side chains and TMS end-capping as well as porous graphitic carbon resins (PGC) have recently been introduced, combined with conventional reversed-phase solvents (Alley et al., 2009; Cavaliere et al., 2021; Cerrato et al., 2021; Montone et al., 2021) (Fig. 4). Similarly, zwitterionic, diol, amide, amino and silica hydrophilic interaction liquid chromatography (HILIC) procedures have been recently optimized for peptidomic analysis of short peptides (Spicer & Krokhin, 2018); they use hydrophilic stationary phases interacting with charged residues in peptides, and acetonitrile and gradients of water as typical eluents including volatile salts (Piovesana et al., 2020). Very recently, chromatographic runs through dedicated chromatographic systems connecting simultaneously in parallel conventional C18- and PGC-based columns have allowed to obtain deeper peptidomic profiles and concomitant, comprehensive characterizations of medium to large-size and small peptides, respectively (Jennings et al., 2021).
Methodologies for extraction and separation of short-chain bioactive peptides
2021, Biologically Active Peptides: From Basic Science to Applications for Human Health