Peptide biomarkers for identifying the species origin of gelatin using coupled UPLC-MS/MS

Abstract

Liquid chromatography linked with mass spectrometry (LC-MS) was used to analyse gelatin from four different species after a trypsin digest. Using chemometric software to analyse the data it was possible to find peptide fragments that were specific to each species of gelatin: porcine, bovine, chicken or fish. Identification of these peptides was challenging due to the destructive nature of gelatin manufacture. The untargeted workflow method developed allowed identification of 21 unknown gelatin samples with 100% accuracy. Fish gelatin is made from a large range of different species that do not share a common differentiating protein but it was shown that the protein from a parasitic bacteria could be used to identify fish gelatin.

Liquid Chromatography–mass Spectrometry Bottom‐up Proteomic Methods in Animal Species Analysis of Processed Meat for Food Authentication and the Detection of Adulterations

“…The marker should be: (1) resistant to thermal processing (peptide should be consistently present after heat treatment across all replicates); (2) unique or without similarity to related species (peptide ions should be crosschecked in the chromatogram of the other meat species and only the peptides that are present in one meat type should be selected); (3) easily detected with LC-MS/MS (high abundance, good signal to noise ratio at low concentration); (4) having length between 6 and 25 amino acid residues; (5) having trypsin specific cleavage sites at both ends, and no missed cleavages; (6) abundant in muscle; (7) reproducible (being extracted and digested every time) (Sarah et al, 2016;Li et al, 2018;Pan et al, 2018;Prandi et al, 2019). Endopeptidase trypsin, which is commonly used for digestion, cleaves the protein chain at the C-terminal side of lysine (K) and arginine (R) residues (Ward et al, 2018). However, in some cases, unique peptides derived from protein fragmentation or nonspecific digestion were also identified and reported.…”

“…They have demonstrated a new method based on shotgun spectral matching of peptide mass spectra, to identify 16 mammalian and 10 bird species in raw and processed samples using LC-MS/MS. There are two proteomic approaches used for species speciation (Ward et al, 2018). One of them is a comprehensive "shotgun" approach and the other a targeted method.…”

“…The coupling of effective liquid chromatography separation with selective mass spectrometry detection provides a rapid, stable, sensitive, specific, and high-throughput analytical tool for the identification and quantification of peptide markers in food authenticity. The popularity of LC-MS/MS methods is confirmed by the increasing number of publications dedicated to the applications of high-resolution mass spectrometry, and targeted methods using MRM for identification and quantification of species-specific peptides in a variety of raw and processed food samples such as; meat (Von Bargen et al, 2014;Ruiz Orduna et al, 2015;Sarah et al, 2016), fish (Carrera et al, 2012), bread (Korte et al, 2018), cookies (Korte et al, 2018), milk (Chen et al, 2016), gelatin (Ward et al, 2018), shrimps (Hu et al, 2018), and also in different samples such as bones (Marbaix et al, 2016), blood (Lecrenier et al, 2016), urine (Gallien et al, 2012), leather goods (Izuchi et al, 2016), or even glue (Kumazawa et al, 2018). Taking into consideration meat samples, there are many examples of using a protein based LC-MS/MS approach to determine species adulterations not only for processed samples but more often for raw meat products.…”

Graphitized Carbon Black Enrichment and UHPLC-MS/MS Allow to Meet the Challenge of Small Chain Peptidomics in Urine

“…In this context, one of the most developed research fields is the discovery of new bioactive peptides in food or food byproducts with health-promoting bioactivities, such as antioxidant, antihypertensive, and antimicrobial properties; 1−4 short peptides can be investigated in food also as biomarkers for food traceability. 5 Another emerging and promising field is the identification of short peptide biomarkers of disease 6 because peptides can have specific functions, for instance, in modulating cancer cells aggressiveness. 7 In the latter context, while peptide sequences longer than 4 amino acids can be investigated by application of proteomics technologies, small peptides (i.e., dipeptides, tripeptides, and tetrapeptides) cannot be dealt with in such a way and are currently mainly investigated within untargeted metabolomics studies.…”

“…Short peptide sequences are emerging as promising analytes in different research fields. In this context, one of the most developed research fields is the discovery of new bioactive peptides in food or food byproducts with health-promoting bioactivities, such as antioxidant, antihypertensive, and antimicrobial properties; − short peptides can be investigated in food also as biomarkers for food traceability . Another emerging and promising field is the identification of short peptide biomarkers of disease because peptides can have specific functions, for instance, in modulating cancer cells aggressiveness .…”