Modern proteomic methodologies for the characterization of lactosylation protein targets in milk

Abstract

Heat treatment of milk induces the Maillard reaction between lactose and proteins; in this context, β-lactoglobulin and α-lactalbumin adducts have been used as markers to monitor milk quality. Since some milk proteins have been reported as essential for the delivery of microelements and, being resistant against proteolysis in the gastrointestinal tract, also contributing to the acquired immune response against pathogens and the stimulation of cellular proliferation, it is crucial to systematically determine the milk subproteome affected by the Maillard reaction for a careful evaluation of aliment functional properties. This is more important when milk is the unique nutritional source, as in infant diet. To this purpose, a combination of proteomic procedures based on analyte capture by combinatorial peptide ligand libraries, selective trapping of lactosylated peptides by m-aminophenylboronic acid-agarose chromatography and collision-induced dissociation and electron transfer dissociation MS was used for systematic identification of the lactosylated proteins in milk samples subjected to different thermal treatments. An exhaustive modification of proteins was observed in milk powdered preparations for infant nutrition. Globally, this approach allowed the identification of 271 non-redundant modification sites in 33 milk proteins, which also included low-abundance components involved in nutrient delivery, defence response against virus/microorganisms and cellular proliferative events. A comparison of the modified peptide identification percentages resulting from electron transfer dissociation or collision-induced dissociation fragmentation spectra confirmed the first activation mode as most advantageous for the analysis of lactosylated proteins. Nutritional, biological and toxicological consequences of these findings are discussed on the basis of the recent literature on this subject, emphasizing their impact on newborn diet.

Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures

“…m-Aminophenylboronic acid-agarose chromatography m-Aminophenylboronic acid-agarose (Sigma) was suspended in washing buffer (250 mM ammonium acetate, 50 mM MgCl 2 , pH 8) and packed into a 1 mL polypropylene column (85 × 7.5 mm) (Qiagen, Germany) [37]. The column was equilibrated with washing buffer before tryptic digest (600 μg), dissolved in washing buffer (200 μL), was loaded at 4°C.…”

“…Digests were analyzed by nanoLC-ESI-LIT-MS/MS using a LTQ XL mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with a Proxeon nanospray source connected to an Easy-nanoLC (Proxeon, Odense, Denmark) [37,58]. Peptide mixtures were separated on an Easy C 18 column (10× 0.075 mm, 3 mm) (Proxeon, Odense, Denmark) using a gradient of acetonitrile containing 0.1% formic acid in aqueous 0.1% formic acid; acetonitrile ramped from 5% to 40% over 90 min, from 40% to 60% over 10 min, and from 60% to 95% over 20 min, at a flow rate of 300 nL/min.…”

“…By using a proteomic approach, we recently demonstrated that these and other water-soluble milk proteins (33 in total number) essential for nutrient delivery, defense against pathogens and cellular proliferation become lactosylated during milk processing [37]. Their nature and modification extent have been associated with the harshness of product manufacturing [22][23][24][25][26][27][28][29][30][31][32]37], putatively having possible consequences on protein functional activity, digestibility, allergenic potential and essential amino acid bioavailability. In fact, the Maillard reaction may modify protein residues involved in biocatalysis or affecting polypeptide three-dimensional structure [12,38,39], thus ultimately influencing aliment functional properties.…”

“…β-LGB, α-LAB, αS1-CSN and β-CSN modification sites have been determined by various MS procedures through bottomup experiments [22,24,[32][33][34][35][36]. By using a proteomic approach, we recently demonstrated that these and other water-soluble milk proteins (33 in total number) essential for nutrient delivery, defense against pathogens and cellular proliferation become lactosylated during milk processing [37]. Their nature and modification extent have been associated with the harshness of product manufacturing [22][23][24][25][26][27][28][29][30][31][32]37], putatively having possible consequences on protein functional activity, digestibility, allergenic potential and essential amino acid bioavailability.…”

“…For a careful evaluation of the nutritional and toxicological properties of differently heated milk samples, it is crucial to systematically determine protein subpopulations affected by the Maillard reaction [37,[43][44][45]. This is particularly important for MFG protein components that have recently been identified as responsible for some health-beneficial properties of milk [1,46].…”