Peptide Characterization and Functional Stability of a Partially Hydrolyzed Whey-Based Formula over Time.

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = -0.95; -0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level (p < 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers.

Proteomics of Human Milk: Definition of a Discovery Workflow for Clinical Research Studies.

Milk is a complex biological fluid composed mainly of water, carbohydrates, lipids, proteins, and diverse bioactive factors. Human milk represents a unique tailored source of nutrients that adapts during lactation to the specific needs of the developing infant. Proteins in milk have been studied for decades, and proteomics, peptidomics, and glycoproteomics are the main approaches previously deployed to decipher the proteome of human milk. In the present work, we aimed at implementing a highly automated pipeline for the proteomic analysis of human milk with liquid chromatography mass spectrometry (MS). Commercial human milk samples were used to evaluate and optimize workflows. Centrifugation for defatting milk samples was assessed before and after reduction, alkylation, and enzymatic digestion of proteins, without and with presence of surfactants. Skimmed milk samples were analyzed using isobaric labeling-based quantitative MS on an Orbitrap Tribrid mass spectrometer. Sample fractionation using isoelectric focusing was also evaluated to more deeply profile the human milk proteome. Finally, the most appropriate workflow was transferred to a liquid handling workstation for automated sample preparation. In conclusion, we have defined and describe herein an efficient highly automated proteomic workflow for human milk sample analysis. It is compatible with clinical research, possibly allowing the analysis of sufficiently large cohorts of samples.

Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals, First Action 2018.06.

Background: An acid hydrolysis ultrahigh-performance LC-UV method was evaluated for the determination of total amino acids in infant formula and adult/pediatric nutritional formula. Objective: It was assessed for compliance against AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) established by the Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN). Methods: A single-laboratory validation (SLV) study was conducted as a first step in the process to validate the method. In this SLV, 17 SPIFAN matrices representing a range of infant formula and adult nutritional products were evaluated for their amino acid content. Results: The analytical range was found to be within the needs for all products; some may require a dilution. Evaluation of trueness performed on Standard Reference Material 1849a (Infant/Adult Nutritional Formula) showed all compounds met the SMPR theoretical value, with exceptions for threonine and tyrosine. These may have a bias for the National Institute of Standards and Technology (NIST) data, depending on hydrolysis used in the determination of the NIST certificate of analysis. Conclusions: Based on the results of this SLV, this method met the SMPR and was approved as a First Action method by the AOAC Expert Review Panel on Nutrient Methods on August 28, 2018.

Removal of organochlorines from vegetable oils and its benefits in preventing formation of monochloropropanediol diesters.

This paper reports the first results on depleting certain organochlorines from vegetable oils without the use of any solvent in order to mitigate monochloropropanediol diesters (MCDPE). The concept is based on separating the organochlorines from the bulk oil by using trapping agents (e.g. monoacylglycerols) that can be easily separated from the oil. The process starts by mixing and homogenizing crude vegetable oils with the trapping agent and subsequently separating the trapping agent from the oil bulk via crystallization. The proof-of-concept of the approach is demonstrated on a spiked sunflower model system, solvent extracted crude sunflower oil, industrially produced crude soybean and corn oils. The depletion of organochlorines in the crude oils and its beneficial effect on the MCPDE content in the heat treated samples is measured by LC-MS. The depletion efficacy of the monitored organochlorines was estimated to be in the 60-95 % range. Both the melting point and polarity of the trapping agents affected the depletion efficacy of the organochlorines. Trapping agents with higher melting point and polarity, such as monostearin were more effective in comparison to high melting point but less polar agents such as palm stearin or agents rich in polar but low melting point monolinolein/monoolein. The effect of organochlorine depletion on the subsequent MPCDE levels in heat treated oil was in the range of 60-90 % reduction depending on the type of the studied oil.

Food peptidomics: large scale analysis of small bioactive peptides--a pilot study.

Food peptidomics deals in part with the identification and quantification of nutritionally relevant peptides which are called bioactive peptides. This category of peptides comprises large, medium to small peptides. However, small peptides (2-6 amino acids) represent by far the largest category. Such molecules sit at the interface of both the world of proteomics and small molecule. The purpose of this study was to evaluate the feasibility of developing an LC-MSMS based method to measure such small peptides at a large scale that is representative of the hundreds of known small bioactive peptides. In order to do that we selected a very complex and homogeneous peptide set in terms of chemical and physical properties. This peptide set comprised only di, tri- and tetrapeptides made out of the three branched chain amino acids (valine, leucine and isoleucine). Results showed that at least 60% of these 117 peptides can be uniquely identified although many are isobaric and co-eluting. Moreover, identical results were obtained when spiked into a complex matrix, i.e. hydrolyzed whey protein. In conclusion, these results support the feasibility of a large scale approach and open the door to further development for all potential small bioactive peptides known so far. BIOLOGICAL SIGNIFICANCE: Bioactive peptides are a key category of molecules for functional food application. Most known bioactive peptides are small (less than 5 amino acids) and hence represent a challenge in terms of analysis when using current proteomics techniques. Therefore development of the food peptidomics field through high throughput large scale assays for these molecules is mandatory in the future to better conduct research in this field.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

Imatinib metabolite profiling in parallel to imatinib quantification in plasma of treated patients using liquid chromatography-mass spectrometry.

Besides affecting the systemic bioavailability of the parent drug, drug metabolizing enzymes (DMEs) may produce bioactive and/or toxic metabolites of clinical interest. We have investigated the capability to analyze simultaneously the parent drug and newly identified metabolites in patients' plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The anticancer drug, imatinib, was chosen as a model drug because it has opened a new area in cancer therapy and is given orally and chronically. In addition, resistance and rare but sometimes severe side effects have been reported with this therapy. The quantification of imatinib and the profiling of its metabolites in plasma were established following three steps: (1) set-up of a generic sample extraction and LC-MS/MS conditions, (2) metabolite identification by LC-MS/MS using either in vitro incubations performed with human liver microsomes (HLMs) or patient plasma samples, (3) the simultaneous determination of plasma levels of imatinib and 14 metabolites in the plasma samples of 38 patients. Partial or cross method validation has been done and revealed that precise determinations of metabolite levels can be performed whereas pure standards are not available. Preliminary results indicate that the disposition of imatinib and its metabolites is related to interindividual variables and that outlier metabolite profiles can be revealed. This article underscores that, in addition to usual therapeutic drug monitoring (TDM), LC-MS/MS methods can simultaneously record a complete drug metabolic profile enabling various correlation studies of clinical interest.