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.

Modified peptides as indicators for thermal and nonthermal reactions in processed milk.

Site-specific relative quantification of β-lactoglobulin modifications in heated milk and dairy products was performed to determine their thermal and nonthermal origins and to evaluate marker candidates for milk processing. Therefore, formation kinetics of 19 different structures at 26 binding sites were analyzed by ultrahigh-performance liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (UHPLC-MS/MS/MRM) after specific protein hydrolysis. The results indicate that (i) site-specific analysis of lactulosyllysine may be a more sensitive marker for mild heat treatment than its overall content; (ii) N(ε)-carboxymethyllysine, N-terminal ketoamide, and asparagine deamidation are of thermal origin and may be good markers for rather intensive heat treatment, whereas N(ε)-carboxyethyllysine reflects thermal and nonthermal processes; (iii) the relevance of methylglyoxal-derived arginine modifications is low compared to that of other modifications; (iv) oxidation of methionine and cysteine is a rather weak indicator of thermal impact; and (v) the tryptophan modifications formylkynurenine and kynurenine are of nonthermal origin and further degraded during processing.

Comprehensive analysis of nonenzymatic post-translational β-lactoglobulin modifications in processed milk by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Nonenzymatic post-translational protein modifications (nePTMs) result in changes of the protein structure that may severely influence physiological and technological protein functions. In the present study, ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) was applied for the systematic identification and site-specific analysis of nePTMs of β-lactoglobulin in processed milk. For this purpose, β-lactoglobulin, which had been heated with lactose under conditions to force nePTM formation (7 d/60 °C), was screened for predicted modifications by using full scans and enhanced resolution scan experiments combined with enhanced product ion scans.

Development of a metabolically stable neurotensin receptor 2 (NTS2) ligand.

Subtype-selective neurotensin receptor 2 (NTS2) ligands can be used as molecular probes to investigate the physiological role of neurotensinergic systems and serve as lead compounds to initiate the development of drugs for the treatment of tonic pain. Starting from our recently described NTS2 ligand 1, structural variants of type 2 were synthesized to further improve binding affinity and selectivity to gain metabolic stability. The peptide-peptoid hybrid 2 b showed excellent NTS2 binding affinity (K(i) =2.

Determination of dideoxyosone precursors of AGEs in human lens proteins.

Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins.

Assessment of heat treatment of dairy products by MALDI-TOF-MS.

The formation of the Amadori product from lactose (protein lactosylation) is a major parameter to evaluate the quality of processed milk. Here, MALDI-TOF-MS was used for the relative quantification of lactose-adducts in heated milk. Milk was heated at a temperature of 70, 80, and 100 degrees C between 0 and 300 min, diluted, and subjected directly to MALDI-TOF-MS.

Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products.

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN.

Application of mass spectrometry for the detection of glycation and oxidation products in milk proteins.

Protein mass spectometry techniques, such as electrospray ionization mass spectrometry or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), are effective methods to screen for protein modifications derived from the Maillard reaction. The analysis of the intact proteins reveals the major modification, most commonly the Amadori product, whereas partial enzymatic hydrolysis prior to mass spectrometry additionally allows the detection of minor adducts. Therefore, a mass spectrometric method was developed for the analysis of whey protein modifications occurring during heat treatment.

Analysis of the peptide profile of milk and its changes during thermal treatment and storage.

In this study a new method was developed for analysis of the low molecular weight protein fraction of milk, allowing a simple and fast overview of the peptide profile of various milk samples. For this purpose, immobilized metal affinity chromatography (IMAC) was coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). By this technique, two major peptides in milk could be identified as fragments of alpha-s1-casein.

Site-specific formation of Maillard, oxidation, and condensation products from whey proteins during reaction with lactose.

Heat treatment of dairy products leads to structural changes of proteins, which can severely decrease the nutritional value [Mauron, J. J. Nutr.