Proteomic Approach Based on MALDI-TOF MS To Detect Powdered Milk in Fresh Cow’s Milk

Abstract

Milk and cheese are expensive foodstuffs, and their consumption is spread among the population because of their high nutritional value; for this reason they are often subjected to adulterations. Among the common illegal practices, the addition of powdered derivatives seems very difficult to detect because the adulterant materials have almost the same chemical composition of liquid milk. However, the high temperatures (180-200 °C) used for milk powder production could imply the occurrence of some protein modifications (e.g., glycation, lactosylation, oxidation, deamidation, dehydration). The modified proteins or peptides could then be used as markers for the presence of powdered milk. In this work, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze tryptic digests relevant to samples of raw liquid (without heat treatment), commercial liquid, and powdered cow's milk. Samples were subjected to two-dimensional gel electrophoresis (2-DE); differences among liquid and powder milk were detected at this stage and eventually confirmed by MALDI analysis of the in gel digested proteins. Some diagnostic peptides of powdered milk, attributed to modified whey proteins and/or caseins, were identified. Then, a faster procedure was optimized, consisting of the separation of caseins from milk whey and the subsequent in-solution digestion of the two fractions, with the advantage of obtaining almost the same information in a limited amount of time. Finally, analyses were carried out with the fast procedure on liquid milk samples adulterated with powdered milk at different percentages, and diagnostic peptides were detected down to 1% of adulteration level.

Proteomic characterization of intermediate and advanced glycation end-products in commercial milk samples

“…S2) was compiled and used for MS and MS/MS data-driven database searching. Some of these amino acid modifications have already been detected in milk proteins [2][3][4][5]13,15,17,19,20]; others have been identified in other model proteins/peptides after their heating in the presence of sugars and/or sugar oxidation products [8][9][10]15,[29][30][31][32][33][34][35][36][37][38].…”

“…Regarding milk proteins, peptide mapping experiments were performed on isolated model components heated in vitro with various sugars to simulate food processing conditions or on a very reduced number of proteins isolated by chromatographic or electrophoretic procedures from commercial milk samples [1,4,9,[13][14][15][16][17][18] and references therein]. Although a number of studies was realized in this context, AGEs assignment was generally restricted to the case of CML adducts, complementing data on oxidized Lys, Met, Cys, Tyr and Trp derivatives, lysinoalanine, histidinoalanine, pyroglutamate and deamidated Asn [2][3][4]13,19]. Recently, untargeted LC-ESI-MS/MS multiple reaction monitoring (MRM) procedures were used to evaluate the different AGEs present in β-lactoglobulin (β-LG) from heated milk models [19].…”

The role of direct high-resolution mass spectrometry in foodomics

“…Among other applications, it has been used to determine the origin of food [69][70][71] and food adulterations [72][73][74][75][76][77]. The discrimination of samples on the basis of their protein fingerprints using MALDI-MS is also an intense area of research [78][79][80].…”

“…By using the whole spectrum of peaks obtained from the analysis, MALDI-TOF MS showed good potential for rapid detection of milk adulteration. Similarly the detection of fraudulent addition of cow milk to sheep and goat milks (of higher value) was investigated by MALDI-TOF MS [75]. Lipid profiles for the analyzed milks showed a different abundance ratio of the ions m/z 703 and 706 which was directly correlated with addition of cow milk into sheep or goat milks.…”

Advanced Mass Spectrometry

“…Proteomic approach to detect powdered milk in fresh cow's milk [114] MALDI-TOF-TOF Proteins as targets of ferrous-catalyzed oxidation: identification of protein carbonyls [37] LC-TOF Evaluation of the immunostimulating activity of soybeans via the determination of the functional peptide soymetide [115] SELDI-TOF Effect of NaCl and sucrose tastants on protein composition of oral fluid [44] MALDI-TOF Hydrolysis of β-lactoglobulin by trypsin under acidic pH [116] UHPLC-TOF Quantification of Se-methylselenocysteine and its γ-glutamyl derivative from naturally Se-enriched green bean (Phaseolus vulgaris vulgaris) [50] MALDI-TOF Detection of sheep and goat milk adulterations by direct MALDI-TOF-MS analysis of milk tryptic digests [117] CE-TOF Identification of bioactive peptides in hypoallergenic infant milk formulas [58] UHPLC-QqTOF Differentiation bovine and porcine gelatins in food products [118] [120]…”