Reduction of multiple reaction monitoring protein target list using correlation analysisa.

High mass resolution mass spectrometry provides hundreds to thousands of protein identifications per sample, and quantification is typically performed using label-free quantification. However, the gold standard of quantitative proteomics is multiple reaction monitoring (MRM) using triple quadrupole mass spectrometers and stable isotope reference peptides. This raises the question how to reduce a large data set to a small one without losing essential information. Here we present the reduction of such a data set using correlation analysis of bovine dairy ingredients and derived products. We were able to explain the variance in the proteomics data set using only 9 proteins across all major dairy protein classes: caseins, whey, and milk fat globule membrane proteins. We term this method Trinity-MRM. The reproducibility of the protein extraction and Trinity-MRM methods was shown to be below 5% in independent experiments (multi-day single-user and single-day multi-user) using double cream. Further application of this reductionist approach might include screening of large sample cohorts for biologically interesting samples before analysis by high-resolution mass spectrometry or other omics methodologies.

Protein levels and protease activity in milk from mothers of pre-term infants: A prospective longitudinal study of human milk macronutrient composition.

BACKGROUND AND AIMS: The composition and enzymology of human milk changes throughout the lactation period, and differ for mothers who give birth prematurely compared to those who deliver at full-term. Understanding the composition of milk from mothers of very low birth weight premature infants is of great significance, and the objective of this study was to evaluate the composition, protein profile and plasmin activity of milk from mothers who delivered infants at different gestational ages. METHODS: Samples of human milk were donated by women (n = 74) in the Cork, Ireland, area who gave birth to full-term (>37 weeks gestation, FT), pre-term (32-37 weeks, PT) and very pre-term (≤32 weeks, VPT) infants. FT milk was collected at 1, 3, 6 and 10 weeks post-partum (PP), while PT and VPT milk was collected weekly until the FT due date of the infant and subsequently followed the FT protocol. RESULTS: Gestational age did not significantly affect lactose or fat content or total energy content of milk. However, protein content, and levels of some individual proteins, were significantly affected by both gestational age at birth and duration of lactation, with significantly higher protein levels in PT or VPT milk samples at 0-7 days and 1-2 months, respectively. Plasmin activity was significantly higher in VPT milk, indicating differences in proteolytic processing in milk. CONCLUSION: Compositional differences between the milk of mothers of term and pre-term infants were greatest in terms of the protein profile, which showed both qualitative and quantitative differences, as well as difference in proteolytic activity.

Longitudinal Analysis of Macronutrient Composition in Preterm and Term Human Milk: A Prospective Cohort Study.

BACKGROUND: Mother's own milk is the optimal source of nutrients and provides numerous health advantages for mothers and infants. As they have supplementary nutritional needs, very preterm infants may require fortification of human milk (HM). Addressing HM composition and variations is essential to optimize HM fortification strategies for these vulnerable infants. AIMS: To analyze and compare macronutrient composition in HM of mothers lactating very preterm (PT) (28 0/7 to 32 6/7 weeks of gestational age, GA) and term (T) infants (37 0/7 to 41 6/7 weeks of GA) over time, both at similar postnatal and postmenstrual ages, and to investigate other potential factors of variations. METHODS: Milk samples from 27 mothers of the PT infants and 34 mothers of the T infants were collected longitudinally at 12 points in time during four months for the PT HM and eight points in time during two months for the T HM. Macronutrient composition (proteins, fat, and lactose) and energy were measured using a mid-infrared milk analyzer, corrected by bicinchoninic acid (BCA) assay for total protein content. RESULTS: Analysis of 500 HM samples revealed large inter- and intra-subject variations in both groups. Proteins decreased from birth to four months in the PT and the T HM without significant differences at any postnatal time point, while it was lower around term equivalent age in PT HM. Lactose content remained stable and comparable over time. The PT HM contained significantly more fat and tended to be more caloric in the first two weeks of lactation, while the T HM revealed higher fat and higher energy content later during lactation (three to eight weeks). In both groups, male gender was associated with more fat and energy content. The gender association was stronger in the PT group, and it remained significant after adjustments. CONCLUSION: Longitudinal measurements of macronutrients compositions of the PT and the T HM showed only small differences at similar postnatal stages in our population. However, numerous differences exist at similar postmenstrual ages. Male gender seems to be associated with a higher content in fat, especially in the PT HM. This study provides original information on macronutrient composition and variations of HM, which is important to consider for the optimization of nutrition and growth of PT infants.

Vitamin E and vitamin E acetate absorption from self-assembly systems under pancreas insufficiency conditions.

We determined the bioavailability of vitamin E from self-assembly structures in patients with diagnosed chronic pancreas insufficiency. Vitamin E solubilized in dispersed inverted bicontinuous cubic phase and in micellar formulation was delivered directly to the small intestine by tube-feeding. A cross-over study with randomization of 6 subjects and 2 treatments including a combined dose of 18 mg (27 IU) of vitamin E (RRR-[5,7-methyl-((2)H6)]-α-tocopherol) and 27 mg (27 IU) vitamin E acetate (RRR-[5-methyl-(2)H3]-α-tocopheryl acetate) was applied over a time period of 1 h. Plasma samples were collected for 56 h and analyzed by liquid chromatography-mass spectrometry. Appearance of labeled tocopherols originating from the treatment started at 25 h and reached Cmax (0.6-4.6 µM depending on subject) in the 7-9 h window. From the Tmax onwards, both forms of tocopherols diminished slowly to 30-50% of their maxima within 56 h. Strong inter-individual variation was observed in the plasma appearance curves (relative standard deviation varied between 38-45%). No significant discrimination was found between the absorption of free or acetylated forms of deuterated α-tocopherol confirming that application of acetylated α-tocopherol provides the same bioavailability as free α-tocopherol. This observation is valid in both dispersed inverted bicontinuous cubic phase and micellar formulations. Furthermore, since the area-under-the-curve values from cubic phase and from micellar formulations are similar, the cubic phase formulation could represent an alternative delivery system for lipophilic micronutrients in conditions or studies where polysorbate-based micelles cannot be generated.

Non-covalent binding of proteins to polyphenols correlates with their amino acid sequence.

The present paper describes the assessment of non-covalent binding (NCB) between milk proteins and polyphenols and its correlation with the physicochemical parameters of proteins. A method based on ultrafiltration and liquid chromatography-tandem mass spectrometry was used to analyse free and non-covalently bound polyphenols (ligands) in mixtures with major milk proteins. Binding strength values of individual polyphenols were normalised to those obtained with quercitrin (quercetin-3-O-rhamnoside), used as a reference compound. NCB data acquired by experiments at pH 6.6 without any preliminary protein denaturation were correlated with the physicochemical parameters of ligands and proteins. Unsupervised multivariate analysis revealed that NCB of proteins clustered according to their family (caseins separated from albumins). Based on this model, a predictive relationship was observed between protein-polyphenol binding strength and primary/secondary structure parameters of the proteins e.g. number of charges, proline residues and extended strand. These results confirm that, under the investigated experimental conditions, the NCB between polyphenols and protein mixtures can be predicted and optimised based on the molecular structures.

Comprehensive analysis of vitamin E constituents in human plasma by liquid chromatography-mass spectrometry.

The present paper describes the development and validation of a normal-phase liquid chromatography-mass spectrometry (NP-HPLC-MS) method for the screening and quantification of vitamin E constituents in human plasma and food matrixes. Liquid-liquid extraction combined with isotope dilution was applied to extract the lipophilic target analytes. Baseline separation of alpha-tocopherylacetate, alpha-tocopherol, alpha-tocotrienol, alpha-tocopherylquinone, beta-tocopherol, gamma-tocopherol, beta-tocotrienol, gamma-tocotrienol, delta-tocopherol, and delta-tocotrienol was achieved utilizing a normal-phase amine column operated with n-hexane and 1,4-dioxane as solvents. Detection was achieved by positive-ion atmospheric-pressure chemical ionization (APCI). Key features of the method are lower limits of detection, 3-51 nmoles/L; lower limits of quantification, 8-168 nmoles/L; linearity coefficients, 0.9778-0.9989; linear ranges, 0.01-29 micromol/L; recoveries, 53-92%; accuracies, 99-103%; intraday precisions, 2-17%; interday precisions, 5-18%; and suppression values, 0-29%. Fragmentation of tocopherols was studied by tandem mass spectrometry, and a fragmentation scheme for tocotrienols/tocopherols is postulated. Neutral-loss and precursor-ion scan experiments were performed for targeted discovery of oxidation products of tocopherols in human blood and fish oil, the latter being an important food component. The presented data suggest that this method will help to expand the number of quantified/discovered vitamin E constituents detected in food products and analyzed during human/animal trials in order to give a more comprehensive picture to nutritionists about the fate of vitamin E.

Proteomics of the rat gut: analysis of the myenteric plexus-longitudinal muscle preparation.

The enteric nervous system (ENS)--present all along the gastrointestinal tract - is the largest and most complicated division of the peripheral nervous system that can function independently of the brain. The peripheral nerve cells are organized in two separate but interconnected meshworks, called the myenteric and submucous plexus. The nervous control of intestinal motility is primarily governed by the myenteric plexus (MP), which lies in-between the longitudinal- (LM) and circular-muscle layers and regulates their functions. To determine whether the proteomic technology is adapted to the analysis of specific gut tissues, we dissected the MP-LM layers from the jejunum, ileum, and colon of Long Evans rats, homogenized them, and separated the proteins using two-dimensional gel electrophoresis. A subset of all the visualized protein spots, covering the entire range of molecular weights and isoelectric points, was then selected and further analyzed by matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry. We identified around 80 proteins in each gut segment, and among those, five were segment-specific. Most of the proteins identified were derived from muscle cells, but we also detected some neuron-specific proteins. This study represents, to our knowledge, the first extensive protein catalog of a neuromuscular layer of the rat intestine and it may constitute the basis to understand pathophysiological mechanisms related to the ENS.

Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes.

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent (12.2 +/- 0.2 and 7.5 +/- 0.2%, respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.