Untargeted Proteomics-Based Profiling for the Identification of Novel Processing-Induced Protein Modifications in Milk.

Nonenzymatic post-translational protein modifications (nePTMs) affect the nutritional, physiological, and technological properties of proteins in food and in vivo. In contrast to the usual targeted analyses, the present study determined nePTMs in processed milk in a truly untargeted proteomic approach. Thus, it was possible to determine to which extent known nePTM structures explain protein modifications in processed milk and to detect and identify novel products. The method combined ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with bioinformatic data analysis by the software XCMS. The nePTMs detected by untargeted profiling of a ß-lactoglobulin-lactose model were incorporated in a sensitive scheduled multiple reaction monitoring method to analyze these modifications in milk samples and to monitor their reaction kinetics during thermal treatment. Additionally, we identified the structures of unknown modifications. Lactosylation, carboxymethylation, formylation of lysine and N-terminus, glycation of arginine, oxidation of methionine, tryptophan, and cysteine, oxidative deamination of N-terminus, and deamidation of asparagine and glutamine were the most important reactions of ß-lactoglobulin during milk processing. The isomerization of aspartic acid was observed for the first time in milk products, and N-terminal 4-imidazolidinone was identified as a novel nePTM.

Identification of peptides reflecting the storage of UHT milk by MALDI-TOF-MS peptide profiling.

Proteolysis during the storage of UHT milk is associated with major technological problems, particularly bitter off-flavors and age gelation limiting the shelf life of milk. In this study, untargeted peptide profiling by MALDI-TOF-MS identified peptides that were formed by proteolysis and reflected the storage of UHT milk. Analysis of nine different commercial UHT samples recorded peptide profiles during and at the end of their shelf life. Relative quantification and sequencing of the peptides revealed that the concentrations of 22 peptides increased significantly during the storage of UHT milk due to the activity of endogenous milk proteases and microbial proteases as well as other unidentified proteolytic mechanisms. Based on highly discriminative AUC values from receiver operator characteristic (ROC) curve analysis, we selected ten peptides as marker candidates. Among those, the peptide ß-casein192-206 (m/z 1668.9) was the most suitable marker differentiating expired-UHT from regular-UHT samples with 100% accuracy. Additionally, ß-casein191-206 (m/z 1782.0) showed 100% specificity and ß-casein139-161 (m/z 2696.4) 100% sensitivity. Thus, ß-casein192-206, either by itself or in combination with ß-casein191-206 and ß-casein139-161, presents a reliable marker to monitor the storage of UHT milk based on proteolytic mechanisms. SIGNIFICANCE: Enzymatic hydrolysis is the main reason why processed milk spoils during storage. The present study recorded peptide profiles to monitor the release or degradation of peptides in stored UHT milk. Among the detected peptides, statistical analysis revealed that the relative concentration of ß-casein192-206 reflected those proteolytic processes most precisely. Food authorities can now refer to ß-casein192-206 as a reliable marker to differentiate between freshly processed milk and products at the end of their shelf life. Furthermore, the food industry can use this marker peptide to improve production processes by monitoring the proteolysis during storage. The recorded peptide profile helps to explain the basic mechanisms leading to storage-induced proteolysis.

Profiling of Multiphosphorylated Peptides in Kefir and Their Release During Simulated Gastrointestinal Digestion.

Casein phosphopeptides are multiphosphorylated milk peptides, which can have anticariogenic activity and improve mineral absorption by binding bivalent metal ions. The present study investigated phosphopeptides in kefir because fermentation may lead to their enhanced release from milk proteins. After selective enrichment by hydroxyapatite extraction, phosphopeptides and their phosphorylation degree were identified by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) before and after enzymatic dephosphorylation. Peptide structures were determined by ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) revealing 27 phosphopeptides in kefir, including nine peptides containing the motif pSpSpSEE, which binds minerals most efficiently. The majority (18) of phosphopeptides were derived from ß-casein, but only three were derived from the most abundant milk protein αs1-casein. After simulated gastrointestinal digestion, MALDI-TOF-MS analysis detected eight putative phosphopeptides in kefir, four of which were assigned by UHPLC-ESI-MS/MS to αs2-casein124-133, αs2-casein137-146, ß-casein30-40, and κ-casein147-161. These results indicate that kefir is a good dietary source of multiphosphorylated peptides.

The German version of the Toronto Structured Interview for Alexithymia: factor structure, reliability, and concurrent validity in a psychiatric patient sample.

BACKGROUND: Recently, the Toronto Structured Interview for Alexithymia (TSIA) was developed to supplement the self-assessment of alexithymia and/or offer a different method of measuring the alexithymia construct. The aim of this study was to evaluate the psychometric properties of a German language translation of the TSIA in a psychiatric patient sample. METHODS: Translation and back-translation were performed until a high agreement of cross-language equivalence was obtained between the German and the original English language version of the TSIA. The TSIA and the German language version of the 20-Item Toronto Alexithymia Scale were administered to 237 psychiatric patients at the departments of psychiatry and psychotherapy in Germany and Switzerland. Videotapes of some of the interviews were recorded for the assessment of interrater reliability. RESULTS: The German version of the TSIA and its 4 scales correlated significantly with the German version of the 20-Item Toronto Alexithymia Scale and its 3 factor scales, providing support for concurrent validity of the interview. Confirmatory factor analyses supported the hierarchical, 4-factor structure obtained with the original English version, with 4 lower-order factors nested within 2 higher-order latent factors. Acceptable levels of internal reliability and interrater reliability were also demonstrated. CONCLUSION: The TSIA is a valid and reliable measure for assessing alexithymia, at least in clinical samples. The TSIA, together with a self-report alexithymia scale, allow for a multimethod approach to assessing alexithymia.