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Background: Human milk oligosaccharides have been shown to relate to the infant gut microbiome. However, the impact of other human milk components on infant gut bacterial colonization remains unexplored., Objectives: Our cross-sectional analysis aimed to investigate associations between human milk components (energy, macronutrients, free amino acids, inflammatory markers, and hormones) and infant gut microbiome diversity and composition (phylum, family, and genus) at 6 mo of age., Methods: Human milk and infant stool samples were collected at 6 mo postpartum. The infant gut microbiome was profiled using 16S rRNA sequencing. Linear regression models were performed to examine associations, adjusting for pregravid BMI (kg/m 2 ), delivery mode, duration of human milk feeding, and infant sex, with q < 0.2 considered significant., Results: This analysis included a total of 54 mothers (100% exclusively feeding human milk) and infants (n = 28 male; 51.9%). Total energy in human milk showed a negative association with α-diversity measures (Chao1 and Shannon). Interleukin (IL)-8 in human milk was positively associated with Chao1 and observed operational taxonomic units. At the family level, human milk glutamine and serine levels showed a negative association with the abundance of Veillonellaceae, whereas isoleucine showed a positive association with Bacteroidaceae. Human milk IL-8 and IL-6 concentrations were positively associated with Bacteroidaceae abundance. IL-8 also had a positive relationship with Bifidobacteriaceae, whereas it had a negative relationship with Streptococcacea and Clostridiaceae. Human milk IL-8 was positively associated with the phylum Bacteroidetes, and negatively associated with Proteobacteria. At the genus level, human milk IL-8 exhibited a positive relationship with Bacteroides, whereas human milk isoleucine had a negative relationship with Bacteroides and Ruminococcus. Pregravid BMI and sex effects were observed., Conclusions: IL-8 in human milk could potentially prepare the infant's immune system to respond effectively to various microorganisms, potentially promoting the growth of beneficial gut bacteria and protecting against pathogens., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Objective: To compare the metabolic profiles of synovial fluid (SF) from patients with anterior cruciate ligament tears and hemarthrosis (HA) with that of normal controls, using 1 H NMR spectroscopy (NMRS)., Methods: Synovial fluid was collected from eleven patients undergoing arthroscopic debridement within 14 days following an anterior cruciate ligament (ACL) tear and hemarthrosis. Ten additional SF samples were obtained from the knees of osteoarthritis-free volunteers to serve as normal controls. The relative concentrations of twenty-eight endogenous SF metabolites (hydroxybutyrate, acetate, acetoacetate, acetone, alanine, arginine, choline, citrate, creatine, creatinine, formate, glucose, glutamate, glutamine, glycerol, glycine, histidine, isoleucine, lactate, leucine, lysine, phenylalanine, proline, pyruvate, threonine, tyrosine, valine, and the mobile components of glycoproteins and lipids) were evaluated using NMRS and quantified using CHENOMX metabolomics analysis software. Mean differences between groups were evaluated with t-tests controlling for multiple comparisons at an overall error rate of 0.10., Results: Statistically significant increases in the levels of glucose, choline, the branched-chain amino acids leucine, isoleucine, and valine, and the mobile components of N-acetyl glycoproteins and lipids were observed in ACL/HA SF as compared with normal controls; lactate levels were reduced., Conclusions: Marked changes occur in the metabolic profiles of human knee fluid following ACL injury and hemarthrosis, suggestive of increased demand and accompanying inflammatory response; potentially increased lipid and glucose metabolism; and possible hyaluronan degradation within the joint following trauma., (Copyright © 2023 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Spodoptera litura and Helicoverpa armigera are polyphagous pests of agricultural crops in the Asian tropics since these pests have been responsible for massive crop and carry economic losses and low commodity production. At the same time, mosquitoes are vectors for numerous dreadful diseases, which is the most important group of insect for their public health concern. Using synthetic insecticides to control the pests can lead to contamination of land surface and groundwater and impact beneficial soil organisms and nontarget species. Applications of bioactive compounds are received considerable attention across the world as alternatives to synthetic insecticides. In the current study, actinobacterial secondary metabolite was isolated from Actinokineospora fastidiosa for the first time. The effect of actinobacterial metabolite (l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester) was assessed on agricultural pest S. litura and H. armigera, mosquito vectors larvae Ae. aegypti, An. stephensi, and Cx. quinquefasciatus. The bioactive fraction was characterized through UV, FTIR, and NMR analysis. GC-MS analyses reveal the existence of a bioactive compound with a respective retention time of 19.740 responsible for larvicidal activity. The bioefficacy of the l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester showed high antifeedant activity on S. litura (80.80%) and H. armigera (84.49%); and larvicidal activity on S. litura (82.77%) and H. armigera (88.00%) at 25 μg/mL concentration, respectively. The effective LC 50 values were 8.07 μg/mL (F = 2.487, r 2  = 0.988, P ≤ 0.05) on S. litura and 7.53 μg/mL (F = 123.25, r 2  = 0.951, P ≤ 0.05) on H. armigera. The mosquito larvicidal effect of isolated compounds l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester treated against Ae. aegypti, An. stephensi, and Cx. quinquefasciatus the obtained percentage mortality was 96.66, 83.24, 64.52, 50.00, and 40.00% against Ae. aegypti; 100.00, 86.22, 73.81, 65.37, and 56.24% against An. stephensi; 100.00, 90.00, 76.24, 68.75, and 56.23% against Cx. quinquefasciatus. The mosquito larvae of Ae. aegypti obtained LC 50 value was 13.25 μg/mL, F = 28.50, r 2  = 0.90; on An. stephensi was 10.19 μg/mL, F = 15.55, r 2  = 0.83, and Cx. quinquefasciatus was 9.68 μg/mL, F = 20.00, r 2  = 0.87. Furthermore, l-isoleucine-, N-allyloxycarbonyl-, and dodecyl ester-treated larvae produced significant pupicidal activity on S. litura (62.71%) and H. armigera (66.50%) at 25 μg/mL, along with increased larval and pupal duration as compared to control group. Treated larvae revealed obliteration in the midgut epithelial cells and destruction of microvilli was noticed as compared to the control. The isolated compounds l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester did not produce any significant mortality on zebrafish embryos in all tested concentrations on biosafety observation. The potential microbial isolated molecule may fit well in IPM programs. Since the risk to human health, the environment, etc. is unknown., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Protein de novo sequencing based on tandem mass spectrometry is a crucial technology that enables the identification of peptides without searching databases and assembling unknown sequence proteins, especially for monoclonal antibodies (mAbs). However, the discrimination of leucine (Leu) and isoleucine (Ile) residues in the target protein sequence is still challenging. Herein, we developed an accurate method by continuous digestion with MS 3 -based fragmentation and multiple spectra integration (evaluated by combined verification score, CVS) to distinguish Leu and Ile residues. Continuous digestion promotes the diversity of peptides in order to expose more Leu and Ile at the N-terminal. CVS integrates multiple MS 3 spectra to reduce the interference from noise and co-fragmented ions and improve accuracy. This method successfully resolved all 75 Leu/Ile in bovine serum albumin, especially 3 consecutive Leu/Ile. We further applied the method to analyze trastuzumab and 67 out of the 68 Leu/Ile from the light chain and heavy chain were accurately discriminated, demonstrating the great potential in mAbs sequencing., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Plum (Prunus spp.) is an economically and nutritionally important stone fruit that is grown worldwide. Gummosis disease (GD) is one of the most common limiting factors that adversely affects the yield and quality of stone fruits such as plum. Elucidating plum fruit metabolomics responses is essential to develop sustainable agricultural practices to combat GD in the future. Herein, an ultra-high-performance liquid chromatography coupled to mass-spectrometry (UHPLC-MS) pseudo-targeted metabolomic profiling was first performed to elucidate the overall metabolic alterations in Asian plum (Prunus salicina Lindl.) fruit in response to GD. The most pivotal differential metabolites, including certain amino acids and proanthocyanidins, in GD and control groups were identified by combining multivariate data analysis with strict statistical criteria. Metabolic pathway enrichment analysis showed that GD induced a series of coordinated defence responses and reprogramming of various metabolic pathways, including glucosinolate biosynthesis, 2-oxocarboxylic acid metabolism, valine, leucine and isoleucine degradation, and isoquinoline alkaloid biosynthesis pathways. Using UHPLC-MS-based pseudo-targeted metabolomic profiling, we systematically evaluated overall metabolic modifications in Asian plum fruits in response to GD for the first time. The identified metabolic pathway alterations helped to better understand the internal relationships and related metabolic networks.

This study aimed to determine the association between the seed coat color of two chia seed genotypes for their composition, protein content, amino acid, and fatty acid profiles. The optimal pH for protein isolation for both genotypes (BCPI and WCPI) was 10, based on protein purity and solubility. Fatty acid profiling indicated, overall, 18 different fatty acids higher in BCPI10 with linolenic acid domination (∼66%) followed by linoleic acid (∼19%) and oleic acid (∼6%), contributing PUFAs (∼86%). Optimized protein isolates, black (BCPI10) and white (WCPI10) chia, had shown purity, L*-value, solubility, and yields of 90.65%, 75.86%, 77.75%, 11.30%, and 90.00%, 77.83%, 76.07%, 10.69%, respectively. BCPI10 depicted higher EAA (33.19 g/100 g N) and EEA indices (57.676%) compared to WCPI10 (32.14 g/100 g N) and 56.360%, respectively. Amino acid profiling indicated higher, PER, TAA, TEAA, TNEAA, TAAA, TBA, acidic AA values for BCPI10, and higher leucine/isoleucine ratio for WCPI10 having leucine and sulfur amino acids as limiting amino acids. BCPI10 had higher sulfur-containing amino acid contents, as the main contributor to the albumin a water-soluble fraction, leading to its higher in vitro digestibility (71.97%) than WCPI10 (67.70%). Both isolates exhibited good WHC and OHC of 3.18, 2.39 and 3.00, 2.20, respectively. Both protein isolates had similar ∆Td (°C) values with some variation in FTIR spectrum from 1000 cm -1 to 1651 cm -1 having more peak intensity for BCPI10. SDS-PAGE indicated bands at 150 kDa, representing globulin and mild bands at 25-33 kDa for glutelin and albumin. A significant (p < 0.05) variation reported in this study for protein and lipid profiles of both genotype attributes to genetic differences between the seeds. PRACTICAL APPLICATION: Based on the nutritional profile, both chia seed isolates (black and white) are suitable for consumption with an edge for black seed when supplemented with their limiting amino acids. The high values of the functional properties and structural characteristics combined with high nutritional values make the chia protein isolate an excellent source of raw material for various food formulations. Fatty acid profile of the oils from the genotypes showed the presence of high amounts of unsaturated fatty acids, especially the PUFAs with more number of fatty acids in black chia seed. The excellent lipid profile of chia seed oil indicates the benefit of using chia seed oil as a source of essential fatty acids in the human diet for optimal health., (© 2022 Institute of Food Technologists®.)

Background: Branched-chain amino acids (BCAAs: isoleucine, leucine, and valine) and aromatic amino acids (AAAs: phenylalanine and tyrosine) are hypothesized to influence early-life obesity risk., Objective: To assess HM free amino acid (AA) concentrations and infant intakes of HM AAs from women with obesity (OB) compared to those with normal weight (NW) and determine the relationships between HM AA consumption and infant growth., Methods: HM samples were collected at 0.5 (n = 151), 2 (n = 129), and 6 (n = 93) months postpartum from mothers with NW (body mass index [BMI] = 18.5-24.9 kg/m 2 ) and OB (BMI > 30 kg/m 2 ). HM AAs were quantified via mass spectrometry. Infant HM intake, anthropometrics and body composition were assessed. Linear mixed-effects models (LMEM) examined the relationships between maternal BMI and HM AA intakes, and HM AA intake and infant growth over the first 6 months postpartum after adjusting for maternal and infant characteristics., Results: Maternal BMI was positively associated with infant intakes of isoleucine, leucine, and AAAs across timepoints. HM AA intakes were positively associated with weight-for-length z-score, fat mass index, and fat-free mass index in infants (p < 0.05)., Conclusions: Maternal BMI led to differences in HM AA composition, which was associated with infant body composition., (© 2021 World Obesity Federation.)

In this article, we investigate the effects of the isoleucine (ILE) N amino acid chain growth, N = 1.0.6, the ILE conformational effect as well as the solvent presence on the electrical and magnetic spectroscopic properties when these compounds are in aqueous solution. Computational molecular dynamics simulations were performed to include the solvent medium and generate uncorrelated configurations involving solute-solvent structures. The charge point model for solvent was used to obtain the results for quantum mechanical calculation, in special DFT calculations, for (ILE) N structures. Our results for the magnetic shielding constant obtained via GIAO-DFT-NMR calculations show that there is evidence of a magnetic behavior that characterizes the number of peptide bonds and, therefore, how the N isoleucine polypeptide chain is composed. TD-DFT results also show an absorption band shift to larger wavelengths indicating a dependence on N growth., (© 2020 Wiley Periodicals LLC.)

Herein we propose, for the first time, a rapid method based on flow injection analysis, electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) and multivariate calibration for the determination of l-leucine, l-isoleucine and L-allo-isoleucine in saliva. As far as we know, multivariate calibration has never been applied to the data from this non-separative approach. The possibilities of its use were explored and the results obtained were compared with the corresponding ones when using univariate calibration. Partial least square regression (PLS1) multivariate calibration models were built for each analyte by analyzing different saliva samples, and were subsequently applied to the analysis of another set of samples which had not been used in any calibration step. For Leu, the model worked satisfactorily with root mean square errors in the prediction step of 17%. This error can be considered acceptable and is common in methodologies that do not include a separation step. Results were compared with those obtained when univariate calibration was used, using the m/z transition 132.1 → 43.0 as the quantitation variable. In this case, the obtained results were not acceptable, with RMSEP of 236%, due to the fact that saliva samples contained another compound, different to the target analytes, which also shared the same transition. Ile and aIle have the same fragmentation patterns, so quantification of the sum of both compounds was performed, with RMSEP of 14% using a PLS1 model. Similar results were obtained when a univariate calibration model using the m/z transition 132.1 → 69.0 was employed. However, the use of this transition should be carefully examined when other compounds present in the matrix contribute to the analytical signal. The method increases sample throughput more than one order of magnitude compared to the corresponding LC-ESI-MS/MS method and is especially suitable as screening. When abnormally high or low concentrations of the analytes studied are obtained, the use of the method that includes separation is recommended to confirm the results., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The molecular recognition ability of tryptophan (Trp) for isomeric amino acids, such as leucine (Leu) and isoleucine (Ile), and isomeric amino acid-containing dipeptides, such as Leu-Gly, Ile-Gly, Gly-Leu, and Gly-Ile (where Gly denotes glycine), was investigated using a tandem mass spectrometer equipped with an electrospray ionization source and cold ion trap. The ultraviolet photodissociation spectra of the cold gas-phase clusters of Leu and Ile with Na + Trp in the wavelength range of 265-290 nm revealed that the relative intensities of Leu and Ile were only different in the wavelength range of 265-273 nm; however, no differences in the relative intensities were observed when the wavelength exceeded 274 nm. The molecular recognition ability of photoexcited Trp was used for the identification and quantification of Leu and Ile in dipeptides in solution. The mole fractions of Leu and Ile in dipeptides could be determined from the abundances observed in a single product ion spectrum of the cold gas-phase clusters of dipeptides with Na + Trp.

Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of E. coli between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (K d ) of 63 ± 6 μM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM-1493 μM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.

Chemical shifts are often the only nuclear magnetic resonance parameter that can be obtained for challenging macromolecular systems. Here we present a framework to derive the conformational sampling of isoleucine side chains from 13 C chemical shifts and demonstrate that side-chain conformations in a low-populated folding intermediate can be determined.

Background: This study evaluates the effects of training (on running distance measured with a Cooper test) in 3 weeks in non-professional athletes using PycnoRacer®, a fitness drink (FD) including Pycnogenol® during the training period., Methods: Pycnogenol® has been used in preclinical conditions and prevention. PycnoRacer® is a liquid combination of Pycnogenol® (15 mg), L-leucine (0.6 g), L-arginine (0.3 g), L-isoleucine (0.3 g), and L-valine (0.3 g). Two comparable groups (one using the FD) were formed: 60 training athletes were requested to drink one bottle 4 times/day, while 65 controls did not use PycnoRacer® or other comparable sports drinks. All subjects had a strong athletic background and advanced knowledge of the procedures of the tests. Two daily training sessions were completed. The sessions consisted in warming up and running a Cooper test., Results: 125 subjects completed the three weeks according to plans. There were nine dropouts due to logistical or working problems. Subjects using the FD improved on average by 18.83% (range 12-23%) in their running distance with training. The difference with controls was significant (P<0.05) at 3 weeks (controls improved on average by 8.9%; range 3-17.4%). The difference between the two groups was on average 9.93% (P<0.05). A comparable increase in VO2max was observed in the groups. In the FD group the increase was on average by 10.05 mL/kg/min compared to 4.95 mL/kg/min in controls, with a difference of 5.1 mL/kg/min (P<0.05). A VAS score showed comparable values. Lower values (concerning muscular pain and cramps) were observed in FD subjects (P<0.05) at the end of the 3 weeks of training. The level of plasma free radicals (PFR) values after the last Cooper test was significantly lower at 3 weeks in the FD group (P<0.05). No intolerance problem was observed by subjects using the FD., Conclusions: In conclusion, the use of PycnoRacer® improved training, running distance, VO2max and PFR decreasing muscular pain and cramps.

Objectives: An important aspect of bipolar disorder (BD) research is the identification of biomarkers pertaining to the somatic health state. The branched-chain essential amino acids (BCAAs), viz valine, leucine and isoleucine, have been proposed as biomarkers of an individual's health state, given their influence on protein synthesis and gluconeogenesis inhibition. Methods: BCAA levels of 141 euthymic/subsyndromal individuals with BD and 141 matched healthy controls (HC) were analysed by high-pressure lipid chromatography and correlated with clinical psychiatric, anthropometric and metabolic parameters. Results: BD and HC did not differ in valine and isoleucine, whereas leucine was significantly lower in BD. Furthermore, correlations were found between BCAAs and anthropometric and glucose metabolism data. All BCAAs correlated with lipid metabolism parameters in females. There were no associations between BCAAs and long-term clinical parameters of BD. A negative correlation was found between valine and Hamilton Depression-Scale, and Beck Depression Inventory II, in male individuals Conclusions: Our results indicate the utility of BCAAs as biomarkers for the current state of health, also in BD. As BD individuals have a high risk for overweight/obesity, in association with comorbid medical conditions (e.g. cardiovascular diseases or insulin resistance), health state markers are urgently required. However, no illness-specific associations were found in this euthymic/subsyndromal BD group.

Phytochemical defense responses of plants are often herbivore-specific and can be affected by a herbivore's feeding mode. However, comprehensive studies documenting the impact of multiple herbivores from different feeding guilds on induced phytochemical responses in distal leaves and its consequences for plant-mediated herbivore interactions are limited and findings are inconsistent. We investigated how herbivory by leaf-chewing caterpillars, cell-content feeding spider mites and phloem-feeding aphids and whiteflies affect secondary metabolomes and phytohormone levels in youngest, non-damaged cotton leaves (distal leaves). Furthermore, bioassays with caterpillars were conducted to assess their performance on distal leaves of plants infested with different herbivores. Caterpillars, and to a lesser degree spider mites, led to a systemic induction of terpenoids with negative consequences for caterpillar performance in the bioassays. Both herbivores reduced levels of various nutrients and potentially antioxidative compounds. Caterpillar damage increased levels of jasmonoyl-L-isoleucine and abscisic acid (ABA), whereas spider mite infestation had no effect on phytohormone levels. Aphid and whitefly infestation did not systemically affect secondary metabolites. Aphids decreased salicylic acid levels while whitefly-infested plants contained increased ABA levels. Neither aphid nor whitefly infestation affected caterpillar performance. In general, feeding mode of a herbivore can affect systemically induced changes in phytochemistry and plant-mediated indirect interactions even though the two phloem-feeding herbivores triggered different phytohormonal responses. The observed reduction of nutrients and potentially antioxidative compounds upon caterpillar and spider mite herbivory underlines the importance of further elucidating the role of resource sequestration as a potential systemic defensive response following herbivory by chewers and cell-content feeding herbivores.

Objectives: L-isoleucine dioxygenase (IDO) specifically transforms L-isoleucine (Ile) to 4-hydroxyisoleucine (4-HIL), and 4-HIL is a promising drug for diabetes. To enhance the activity and catalytic efficiency of IDO, we used directed evolution and site-specific mutagenesis., Results: The IDO gene (ido) derived from Bacillus weihenstephanensis was cloned and expressed in Escherichia coli. Directed evolution using error prone (EP)-PCR and site-specific mutagenesis were conducted. Two improved mutants were obtained after one round of EP-PCR, with Ido N126H exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with Ido T130K showing a 170% increase in activity. Although the activity of the combined mutant Ido N126H/T130K (0.95 ± 0.08 U/mg) was slightly higher than that of the wild-type Ido, its catalytic efficiency was 2.4-fold and 3.0-fold higher than Ido with Ile and α-ketoglutaric acid as substrates. After biotransformation of Ile by E. coli BL21(DE3) expressing Ido N126H/T130K and Ido, 66.50 ± 0.99 mM and 26.09 ± 1.85 mM 4-HIL was synthesized, respectively, in 24 h., Conclusion: Ido N126H/T130K had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.

Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS 3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. Graphical Abstract ᅟ.

Electron-transfer dissociation (ETD) and electron-transfer and higher-energy collision dissociation (EThcD) spectra of short tryptic peptides with leucine/isoleucine residues in neighboring positions demonstrate intensive w-ions. On the contrary, u-ions possess very low intensities (if present at all). Therefore radical site migration is negligible in the applied conditions while ETD (EThcD) spectra allow for the reliable discrimination of the isomeric residues in the sequencing process. The presence of a fragment ion 43.055 mass units lower than z 2 -ion of peptides with IK sequence at their C-termini was shown to be a result of alternative fragmentation starting from the loss of propylammonium ion from the doubly protonated peptide molecule and formation of an oxazole fragment ion.

Lower urinary tract symptoms (LUTS), including urinary incontinence, urgency and nocturia, affect approximately half of women worldwide. Current diagnostic methods for LUTS are invasive and costly, while available treatments are limited by side effects leading to poor patient compliance. In this study, we aimed to identify urine metabolic signatures associated with LUTS using proton nuclear magnetic resonance ( 1 H NMR) spectroscopy. A total of 214 urine samples were collected from women attending tertiary urogynecology clinics (cases; n = 176) and healthy control women attending general gynecology clinics (n = 36). Despite high variation in the urine metabolome across the cohort, associations between urine metabolic profiles and BMI, parity, overactive bladder syndrome, frequency, straining, and bladder storage were identified using KODAMA (knowledge discovery by accuracy maximization). Four distinct urinary metabotypes were identified, one of which was associated with increased urinary frequency and low BMI. Urine from these patients was characterized by increased levels of isoleucine and decreased levels of hippurate. Our study suggests that metabolic profiling of urine samples from LUTS patients offers the potential to identify differences in underlying etiology, which may permit stratification of patient populations and the design of more personalized treatment strategies.

An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination. Graphical Abstract ᅟ.

In this study we investigated 11 isomers with the molecular formula C 6 H 13 NO 2 (m/z 131) to ascertain the potential of utilizing drift tube ion mobility mass spectrometry to aid in the separation of isomeric mixtures. This study of small molecules provides a detailed examination of the application of uniform field ion mobility for a narrow scope of isomers with variations in both bond coordination and stereochemistry. For small molecules, it was observed that in general constitutional isomers are more readily separated by uniform field mobility in comparison to stereoisomers such as enantiomers or diastereomers. Diastereomers exhibited differences in their collision cross section (CCS), but were unresolvable in a mixture, whereas the enantiomers studied did not exhibit statistically different CCS values. A mathematical relationship relating the CCS to resolving power was developed in order to predict the required ion mobility resolving power needed to separate the various isomer classes. For the majority of isomers evaluated in this study, a uniform field-based resolving power of 100 was predicted to be sufficient to resolve over half (∼60%) of all hypothetical isomer pairs, including leucine and isoleucine, whereas their stereoisomers (d- and l-forms) are predicted to be significantly more challenging, if not impossible, to separate by conventional drift tube techniques.

Monoclonal antibodies (mAbs) are large heterogeneous molecules that represent a growing class of therapeutics. De novo sequencing of mAbs becomes necessary when the original cell line or the cDNA is unavailable. An important feature in sequencing of mAbs is the discrimination of isobaric residues (Xle): leucine (Leu) and isoleucine (Ile). An incorrect identification of the Xle site, especially in the complementarity determining regions (CDRs), can result in the production of an antibody with severely compromised efficacy. Multistage fragmentation (MS n ) in the mass spectrometer can provide sufficient evidence for Ile/Leu discrimination. However, most existing methods utilize direct infusion of purified peptides, demanding peptide enrichment which can be labor-intensive and requires large amount of material. Here we introduce an online nano-LCMS n method, which depending on the nature of the peptide, exploits either generation of a signature 69 Da ion from Ile or formation of unique w-ions employing MS 3 (ETD-HCD) for rapid Ile/Leu distinction. This reliable and sensitive method utilizes the Orbitrap Fusion tribid mass spectrometer to rapidly assign multiple Xle residues in the CDRs of mAbs.

The increasing demand for natural foods and beverages, i.e. prepared by excluding synthetic chemicals along the whole production chain, has boosted the adoption of organic and biodynamic cultivation methods which are based on protocols avoiding use of synthetic pesticides. This trend is striking in viticulture, since wine production is largely shaped by the varying drinking attitudes of environment-friendly consumers. Using (1)H NMR, the compositions of grape berries, collected at harvest in 2009 and 2011, in experimental plots cultivated either with biodynamic or organic methods, were compared. Although the analysis provides a comprehensive metabolic profile of berries, the resulting distinctive pattern consists of a few molecules. Lower content of sugars, coumaric and caffeic acids, as well as higher amount of γ-aminobutyric acid (GABA) were observed in biodynamic grapes. The (1)H NMR foodomics approach evidenced a diverse fruit metabolome that could be associated to a different physiological response of plants to the agronomic environment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Despite the great success of mass spectrometry (MS) for de novo protein sequencing, Leu and Ile have been generally considered to be indistinguishable by MS because their molecular masses are exactly the same. Positioning of incorrect Leu/Ile residues in variable domains, especially in CDRs (complementarity determining regions) of an antibody, may result in substantial loss of antigen binding affinity and specificity of the antibody. Here, we describe an integrated LC-MS based strategy, encompassing a combination of HCD (high-energy collisional dissociation) multistage mass spectrometric analysis (HCD-MS n ) and ETD (electron transfer dissociation)-HCD MS 3 analysis using an Orbitrap Fusion mass spectrometer, to reliably identify Leu and Ile residues in proteins and peptides. The merits and limitations of this Leu/Ile discrimination approach are evaluated. Using the new approach, along with proposed decision-making guidelines we unambiguously identified every Leu/Ile residue in peptides containing up to five Leu/Ile residues and molecular masses up to 3000 Da. In addition, we have demonstrated, for the first time, that every Leu/Ile residue in the variable regions of a monoclonal antibody that could not be assigned by antibody germline sequence alignment could be correctly determined using this approach. Our results suggest that, by incorporating this approach into existing de novo antibody sequencing protocols, 100% of antibody amino acid sequences, including identity of Leu and Ile residues, can be accurately obtained solely by means of mass spectrometry. In principle, this integrated, online LC-MS approach for Leu/Ile assignment can be applied to de novo sequencing of any protein or peptide.

We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e., isoleucine, leucine, and valine) and their downstream metabolites (i.e., alanine, glutamine, and glutamate) are important indicators of adipocyte lipogenesis. To perform an analysis, amino acids were sampled using microdialysis, fluorescently labeled in an online reaction, separated using CE, and detected using laser-induced fluorescence (LIF) in a sheath flow cuvette. Separation conditions were optimized for the resolution of the BCAAs isoleucine, leucine, and valine, as well as 13 other amino acids, including ornithine, alanine, glutamine, and glutamate. CE separations were performed in <30 s, and the temporal resolution of the online MD-CE assay was <60 s. Limits of detection (LOD) were 400, 200, and 100 nM for isoleucine, leucine, and valine, respectively. MD-CE dramatically improved throughput in comparison to traditional offline CE methods, allowing 8 replicates of 15 samples (i.e., 120 analyses) to be assayed in <120 min. The MD-CE assay was used to assess the metabolism dynamics of 3T3-L1 cells over time, confirming the utility of the assay.

Numerous nuclear magnetic resonance (NMR) measurements of spin-lattice relaxation times (T1S) for dilute spins such as (13)C have led to investigations of the motional dynamics of individual functional groups in solid materials. In this work, we revisit the Solomon equations and analyze how the heteronuclear cross relaxation between the dilute S (e.g. (13)C) and abundant I (e.g. (1)H) spins affects the measured T1S values in solid-state NMR in the absence of (1)H saturation during the recovery time. It is found theoretically that at the beginning of the S spin magnetization recovery, the existence of non-equilibrium I magnetization introduces the heteronuclear cross relaxation effect onto the recovery of the S spin magnetization and confirmed experimentally that such a heteronuclear cross relaxation effect results in the recovery overshoot phenomena for the dilute spins when T1S is on the same order of T1H, leading to inaccurate measurements of the T1S values. Even when T1S is ten times larger than T1H, the heteronuclear cross relaxation effect on the measured T1S values is still noticeable. Furthermore, this cross relaxation effect on recovery trajectory of the S spins can be manipulated and even suppressed by preparing the initial I and S magnetization, so as to obtain the accurate T1S values. A sample of natural abundance l-isoleucine powder has been used to demonstrate the T1S measurements and their corresponding measured T1C values under various experimental conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly (13)C/(15)N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for (13)C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of (13)C(1)H3-labeled methyls by comparing distance information obtained from three-dimensional methyl-methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl-methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed "NOE-methyl cluster" onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV (13)C(1)H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.

Background: Type 2 diabetes patients (DP) have significantly higher plasma levels of valine, leucine, isoleucine and alanine than the controls. Specific amino acids may acutely and chronically regulate insulin secretion from the pancreatic β-cells. We recently identified a metabolic signature of N-acetyl leucine (Ac-Leu) that strongly predicts diabetes development in mice hair. The Ac-Leu appears to be a potential biomarker candidate related to diabetes. However, the determination of Ac-Leu in human hair has not been reported. We measured the Ac-Leu, and its structure is similar to N-acetyl isoleucine (Ac-Ile) in human hair by ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The developed method was applied to the determination of Ac-Leu and Ac-Ile in the hair of healthy volunteers (HV) and DP., Methods: Ac-Leu, Ac-Ile and N-acetyl norleucine (Ac-Nle, IS) were extracted from human hair samples by a micropulverized extraction procedure, then separated on a C18 column by isocratic elution of acetonitrile-0.1% formic acid in water:0.1% formic acid (14:86, vol./vol.). MRM using the fragmentation transitions of m/z 174.1→86.1 in the positive ESI mode was performed to quantify the N-acetyl leucine, N-acetyl isoleucine and IS., Results: Ac-Leu, Ac-Ile and Ac-Nle in the human hair samples were completely separated by isocratic elution of a 5.0 min duration wash program using a reversed-phase column, and sensitively detected by LC-MS/MS in the ESI(+) MRM mode. The amounts of Ac-Leu and Ac-Ile in the hairs of HV and DP were determined. When comparing the concentrations between DP and those from HV, a statistically significant correlation was observed for the Ac-Leu (p<0.001) and Ac-Ile (p<0.01)., Conclusions: The proposed method is useful for the determination of Ac-Leu and Ac-Ile in the hairs of DP and HV. Human hair may serve as a noninvasive biosample for the diagnosis of diabetes., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively (13)C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The efficient coupling of biotransformation steps to an existing fermentation pathway is an interesting strategy to expand the product portfolio of Corynebacterium glutamicum as whole-cell biocatalyst. This is especially challenging if the biotransformation step comprises a direct link to central metabolism, as in the case of α-ketoglutarate-dependent oxygenase catalysis. Aiming at trans-4-hydroxy-L-proline (Hyp) production from glucose in a minimal medium, the proline-4-hydroxylase gene from Dactylosporangium sp. strain RH1 was introduced into a proline-producing, isoleucine-bradytroph C. glutamicum strain. The production of proline was found to be induced by isoleucine limitation. Proline and Hyp production were found to depend differently on isoleucine limitation. Severe isoleucine limitation was shown to result in proline accumulation and low hydroxylation rates both in batch and continuous cultivation set-ups. The investigation of different steady states with various glucose/isoleucine molar ratios revealed that optimal conditions for Hyp production are met around a molar ratio of 46:1, where isoleucine limitation is sufficient to trigger proline production but the hydroxylation rate is high enough to convert the majority of formed proline to Hyp. A high cell-density fed-batch set-up was designed, capable of producing 7.1 g L(-1) of Hyp from glucose in 23 h with 98.5% conversion of proline to Hyp. Reaction engineering, specifically the fine-tuning of the glucose/isoleucine concentration ratio, enabled control of the fermentation profile and thus the accumulation of the desired product Hyp from glucose in minimal and defined media., (© 2014 Wiley Periodicals, Inc.)

Major depressive disorder (MDD) is a debilitating mood disorder. However, the molecular mechanism(s) underlying depression remain largely unknown. Here, we applied a GC-MS-based metabonomic approach in the chronic unpredictable mild stress (CUMS) model, a well-established rodent model of depression, to investigate significant metabolic changes in the rat prefrontal cortex (PFC). Multivariate statistical analysis - including principal component analysis, partial least squares-discriminate analysis, and pair-wise orthogonal projections to latent structures discriminant - was applied to identify differential PFC metabolites between CUMS rats and healthy controls. As compared to healthy control rats, CUMS rats were characterized by lower levels of isoleucine and glycerol in combination with higher levels of N-acetylaspartate and β-alanine. These findings should provide insight into the pathophysiological mechanism(s) underlying MDD and preliminary leads relevant to diagnostic biomarker discovery for depression., (Copyright © 2014 Elsevier B.V. All rights reserved.)

D-isoleucine (D-Ile) can be assayed using chiral chromatography but the availability of that method is limited by the necessity for special expertise and expensive equipment. We therefore developed a simple and specific colorimetric assay system for D-Ile determination using an artificially created NADP(+)-dependent D-amino acid dehydrogenase (DAADH). The system consists of two reaction steps: the first is the quantitative conversion of D-Ile to (3R)-2-oxo-3-methyl valerate by DAADH in which NADP(+) is converted to NADPH, while the second is chemical conversion of NADPH to reduced water-soluble Tetrazolium-3 via a redox mediator. D-Ile was determined from 1 to 50 µM, and the assay was unaffected by the presence of any of three other isomers (100 µM), alcohol and organic acids.

An efficient approach to easy and reliable differentiation between isomeric leucine and isoleucine in peptide sequencing utilizes multistage electron transfer dissociation and higher energy collision activated dissociation in the Orbitrap Fusion mass spectrometer. The MS(3) method involves production and isolation of primary odd-electron z(•) ions, followed by radical site initiation of their fragmentation with formation of w-ions, characteristic of the isomeric amino acid residues. Six natural nontryptic peptides isolated from the secretion of frog Rana ridibunda were studied. Their lengths were in the range between 15 and 37 amino acids and the number of targeted isomeric (Leu/Ile) residues varied between 1 and 7. The experiments were successful in all 22 cases of Leu/Ile residues, leaving no doubts in identification. The method is extremely selective as the targeted w-ions appear to be the most intense in the spectra. The proposed approach may be incorporated into shotgun proteomics algorithms and allows for the development of an exclusively mass spectrometric method for automated complete de novo sequencing of various peptides and proteins.

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

Improving the amino acid (AA) profile of the diet by using l-Lys, l-Thr, dl-Met, l-Trp and l-Val helps to reduce the dietary CP content, thereby reducing nitrogen excretion while maintaining the performance of pigs. Valine is the fifth limiting AA in cereal-soybean meal-based diets. The extent to which the CP content in the diet can be reduced further without compromising performance depends on the requirement of the next limiting AA. In cereal-soybean meal-based diets, Ile, His and Leu may be the limiting AAs after Val, although information on the requirements for these AAs is scarce. Six experiments were conducted to determine the effect of supplementing a low-CP diet with l-Ile, l-His and l-Leu on the performance of pigs weighing 10 to 20 kg. Experiment 1 was designed to determine the most limiting AA with respect to performance among Ile, His and Leu. A diet 10% deficient in Ile, Leu and His relative to the National Research Council (NRC, 1998) requirement estimates tended to decrease daily feed intake and daily gain by 6% and 8%, respectively. A 10% deficiency in His alone had no effect, whereas a 10% deficiency in Ile or Leu slightly reduced daily feed intake and gain. In the remaining experiments, the standardized ileal digestible (SID) Ile : Lys, His : Lys and Leu : Lys requirements were estimated. In Experiments 2, 3, 4, 5 and 6, 14 blocks of six pigs each were assigned to six levels of SID Ile : Lys (40%, 43%, 46%, 49%, 52% and 55%), His : Lys (20%, 24%, 28%, 32%, 36% and 40%), His : Lys (21%, 24%, 27%, 30%, 33% and 36%), Leu : Lys (70%, 78%, 86%, 94%, 102% and 110%) and Leu : Lys (80%, 90%, 100%, 110%, 120% and 130%), respectively. Across experiments, the estimated SID Ile : Lys, His : Lys and Leu : Lys requirements for maximizing daily gain were 49%, 32% and 102%, respectively, using a curvilinear plateau model. When Ile, His and Leu levels were 10% below the requirement estimate, daily gain was reduced by 9%, 3% and 3%, respectively. The results of this study indicate that the Ile requirement estimate is lower than the current NRC requirement estimate, whereas the Leu and His requirements correspond to those proposed by the NRC.

The distributions of amino acids at most-conserved sites nearest catalytic/active centers (C/AC) in 4,645 sequences of ten enzymes of the glycolytic Embden-Meyerhof-Parnas pathway in Archaea, Bacteria and Eukaryota are similar to the proposed temporal order of their appearance on Earth. Glycine, isoleucine, leucine, valine, glutamic acid and possibly lysine often described as prebiotic, i.e., existing or occurring before the emergence of life, were localized in positional and conservational defined aggregations in all enzymes of all Domains. The distributions of all 20 biologic amino acids in most-conserved sites nearest their C/ACs were quite different either from distributions in sites less-conserved and further from their C/ACs or from all amino acids regardless of their position or conservation. The major concentrations of glycine, e.g., perhaps the earliest prebiotic amino acid, occupies ≈ 16 % of all the most-conserved sites within a volume of ≈ 7-8 Å radius from their C/ACs and decreases linearly towards the molecule's peripheries. Spatially localized major concentrations of isoleucine, leucine and valine are in the mid-conserved and mid-distant sites from their C/ACs in protein interiors. Lysine and glutamic acid comprise ≈ 25-30 % of all amino acids within an irregular volume bounded by ≈ 24-28 Å radii from their C/ACs at the most-distant least-conserved sites. The unreported characteristics of these amino acids: their spatially and conservationally identified concentrations in Archaea, Bacteria and Eukaryota, suggest some common structural organization of glycolytic enzymes that may be relevant to their evolution and that of other proteins. We discuss our data in relation to enzyme evolution, their reported prebiotic putative temporal appearances on Earth, abundances, biological "cost", neighbor-sequence preferences or "ordering" and some thermodynamic parameters.

MALDI in-source decay (ISD) has been used for top-down sequencing of proteins. The use of the matrix 1,5-diaminonapthalene (1,5-DAN) gave abundant w ions, which are formed from the unimolecular dissociation of z• radical fragments via α cleavage reaction and thus help identify which of the isobaric amino acids, Leu or Ile, is present. The high abundance of w ions in MALDI-ISD with 1,5-DAN results from the low collision rate in the MALDI plume. MALDI-ISD with 1,5-DAN appears to be an useful method for the top-down sequencing of proteins, including discrimination of Leu and Ile near the C-terminal end.

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile., (© 2012 Elsevier B.V. All rights reserved.)

Characterization and differentiation of isomers in biological macromolecules using mass spectrometry is one of the most significant challenges facing scientists in the field. The capability of high-resolution MS instruments along with the development of new fragmentation methods now provides the ability to indirectly differentiate between some isomers. This ability has enabled mass spectrometry to evolve into a multidisciplinary technique incorporating areas such as pharmaceutical research, proteomics, polymer science, medicine, environmental chemistry, and recently archeology. This article aims to review recent developments in mass spectrometry methodologies in the identification of structural and spatial isomers in biological macromolecules, such as aspartic acid and isoaspartic acid (Asp/IsoAsp), leucine and isoleucine (Leu/Ile), glutamic acid and γ-glutamic acid, and D/L enantiomers., (Copyright © 2012 Wiley Periodicals, Inc.)

Background & Aims: We previously observed in elderly subjects with Chronic Obstructive Pulmonary Disease (COPD) an enhanced anabolic response to milk protein sip feeding, associated with reduced splanchnic extraction (SPE) of phenylalanine. Milk proteins are known for their high Branched-chain Amino Acids (BCAA) content, but no information is present about splanchnic extraction and metabolism of the individual BCAA in COPD., Objective: To investigate whether BCAA metabolism and SPE of the individual BCAA are altered in COPD during milk protein sip feeding., Design: In elderly subjects with COPD and in healthy age-matched elderly SPE, endogenous rate of appearance (Raendo) of the leucine (LEU), isoleucine (ILE) and valine (VAL) were measured before and during sip feeding of a Whey protein meal. To study the effect of aging, the healthy elderly were compared to a group of healthy young subjects. Stable isotopes of l-[(2)H(3)]-LEU, l-[1-(13)C]-ILE and l-[1-(13)C]-VAL were given on two separate test days orally or intravenously. Simultaneously, l-[ring-(2)H(5)]-phenylalanine (PHE) and l-[ring-(2)H(2)]-tyrosine (TYR) were given to determine the whole body protein breakdown (WbPB), synthesis (WbPS) and NetPS., Results: SPE of all BCAA, TYR, and PHE (p < 0.01) were lower in the COPD group, and the increase in netPS during feeding was higher in the COPD group (P < 0.01) due to higher values for PS (P < 0.001). Raendo of all BCAA, PHE and TYR were higher in the COPD than the healthy elderly group (P < 0.05) before and during feeding (P < 0.001). Sip feeding resulted in a reduction of Raendo of PHE, ILE and VAL (P < 0.05). Postabsorptive Raendo was not different for any of the measured amino acids between the healthy elderly and young group, while sip feeding resulted in a reduction of Raendo of PHE. Only SPE of TYR was higher in the elderly (P < 0.05) and the increase in netPS during sip feeding was independent of aging., Conclusion: The enhanced anabolic response to milk protein sip feeding in normal-weight COPD patients is associated with a reduced splanchnic extraction of multiple amino acids including all branched-chain amino acids. Registration ClinicalTrials.gov = NCT01418469., (Copyright © 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Multiple reaction monitoring (MRM) mass spectrometry may be regarded as the gold standard methodology for quantitative mass spectrometry and has been adopted for the analysis of small molecules especially within the pharmaceutical industry. It can also be applied to the analysis of peptides and proteins and to the measurement of the basic building blocks of proteins, amino acids. Here, we describe the application of MRM mass spectrometry to the measurement of hydroxyproline after acid hydrolysis of various animal tissues. We show that the measurement of hydroxyproline provides an accurate and reliable estimate of the collagen content of such tissues and may be a useful indicator of meat tenderness.

1. Valine (Val) is considered the 4th limiting amino acid for broilers fed on diets containing ingredients from vegetable origin. However, Val and Isoleucine (Ile) may be co-limiting for broilers fed on diets containing animal protein meals. An experiment was conducted to examine growth responses and meat yield of broilers provided diets varying in digestible Val (dVal) and digestible Ile (dIle) concentrations from 28 to 42 d of age. 2. Eight experimental diets varying in dVal (dVal to dLys ratios from 0·66 to 0·76) and dIle (dIle to dLys ratios from 0·57 to 0·67) were studied; digestible Lysine (Lys) was formulated to 9·9 g/kg in all diets. 3. Broilers fed on a negative control (NC) diet supplemented with crystalline Val (L-Val) and crystalline Ile (L-Ile), dVal to dLys = 0·76 and dIle to dLys = 0·67, grew faster and had higher breast meat yield than birds fed on NC + L-Ile (dVal to dLys = 0·66 and dIle to dLys = 0·67), NC + L-Val (dVal to dLys = 0·76 and dIle to dLys = 0·57), and NC + reduced L-Val and L-Ile (dVal to dLys = 0·71 and dIle to dLys = 0·62). 4. Feeding broilers on the NC + L-Val and L-Ile (dVal to dLys = 0·76 and dIle to dLys = 0·67) diets gave similar BW gain, carcase weight and yield and total breast meat weight and yield to birds fed on the positive control-fed broilers with no added L-Val and L-Ile (dVal to dLys = 0·76 and dIle to dLys = 0·67). 5. These results indicate that Val and Ile are co-limiting in diets containing poultry by-product meal.

Many plants used as functional foods or for medicinal purposes have been criticized for their inconsistent physiological effects. Variation in genotype and environmental conditions under which plants are produced can contribute to this inconsistency in biochemical composition. Fenugreek (Trigonella foenum-graecum L.) is a medicinal plant that not only can lower blood glucose and cholesterol levels in animals, but also can be used as a forage crop for livestock feed. Seed content for the bioactive compounds diosgenin, galactomannan and 4-hydroxyisoleucine was characterized for ten fenugreek genotypes under rainfed and irrigated conditions. High and low seed yielding genotype/environment combinations were identified that possessed distinct biochemical and seed production traits. In general high seed yielding genotype/environment combinations exhibited a more stable biochemical composition and consisted largely of irrigated fenugreek. This research indicates that comprehensive biochemical analysis of plant products would facilitate the development of more reliable produce for use by the functional food/medicinal plant industry., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Collisional cooling employed in an orthogonal time-of-flight mass spectrometer (o-TOF MS) stabilizes fragment ions that are generated by matrix-assisted laser desorption ionization in-source decay (MALDI ISD). By variation of the buffer gas pressure, ISD and "post-source decay" (PSD) dissociation channels can be switched on and off to some extent. Under high-pressure conditions, ISD type fragments of post-translationally modified (PTM) peptides are generated that contain even labile bound side groups; the examples of a phosphorylated and an O-glycosylated peptide are shown. At elevated laser fluences, d- and w-type fragment ions of peptides are detected as a result of high-energy side chain cleavage. This allows for differentiation of the isobaric amino acid residues leucine and isoleucine. Reduction of the cooling efficiency by lowering the buffer gas pressure results in the loss of the d-/w-type species, presumably in secondary metastable dissociation processes. This also enhances cleavage of the side groups from the PTM peptides and can be used to corroborate identification of the modification site. Furthermore, these measuring conditions generate small amounts of fragments containing sequence information about the cyclic part of a disulfide peptide by inducing symmetric and asymmetric cleavage of the intramolecular S-S bond.

Most studies concerning the Ile requirement in pigs have been carried out using blood products as a protein source, and these have a relatively low Ile content relative to the other branched-chain AA (BCAA). There are indications that an excess supply of one BCAA can affect the utilization of the other BCAA. Little information is available concerning the Ile requirement in pigs when the supply of the other BCAA is moderate (e.g., in cereal- and soybean meal-based diets). The objective of the present study was to evaluate the response of piglets to Ile supplementation under different nutritional conditions. In all experiments, piglets were housed individually and had ad libitum access to feed during a 3-wk period. The first experiment was carried out to study the response of piglets to an increasing Ile supply by using 2 sources of l-Ile differing in degree of purity. Piglets received either a control diet with 48% standardized ileal digestible (SID) Ile:Lys or 1 of 4 other diets containing graded levels of either source of l-Ile to provide 52 or 56% SID Ile:Lys. All diets were formulated to provide 1.00% SID Lys in the diet. Feed intake and growth were not affected by Ile level or Ile source. Experiment 2 was performed to exclude a possible interaction between Ile and Lys supply. In a 2 x 2 factorial arrangement, 2 levels of Lys (1.00 and 1.15% SID Lys) and 2 levels of Ile (48 and 60% SID Ile:Lys) were used. Growth and G:F were 8 and 7% greater in piglets receiving the diet with the greater Lys content, but the Ile:Lys did not affect performance. No interactions were observed between the Lys and Ile supplies. In Exp. 3, a 2 x 2 factorial arrangement was used to test the effect of protein source (spray-dried blood cells or corn gluten meal) and Ile supply (50 or 65% SID Ile:Lys) on performance in piglets. Both protein sources had an elevated BCAA content but differed in Leu and Val contents. Protein source or Ile supply did not affect feed intake, growth, or G:F in the piglets. Plasma concentrations after an overnight fast reflected the difference in AA concentrations of the diets. In conclusion, the results of these experiments indicate that the SID Ile:Lys requirement may be not greater than 50% in piglets receiving cereal- and soybean meal-based diets with a moderate BCAA content. In contrast to other studies, we could not confirm that the Ile requirement was affected by BCAA content of the diet.

Extensive backbone fragmentation resulting in a-, b-, c-, x-, y- and z-type ions is observed of singly and doubly charged peptide ions through their interaction with a high kinetic energy beam of argon or helium metastable atoms in a modified quadrupole ion trap mass spectrometer. The ability to determine phosphorylation-sites confirms the observation with previous reports and we report the new ability to distinguish between leucine and isoleucine residues and the ability to cleave two covalent bonds of the proline ring resulting in a-, b-, x-, y-, z- and w-type ions. The fragmentation spectra indicate that fragmentation occurs through nonergodic radical ion chemistry akin to electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron ionization dissociation mechanisms. However, metastable atom-activated dissociation mass spectrometry demonstrates three apparent benefits to ECD and ETD: (1) the ability to fragment singly charged precursor ions, (2) the ability to fragment negatively charged ions and (3) the ability to cleave the proline ring that requires the cleavage of two covalent bonds. Helium metastable atoms generated more fragment ions than argon metastable atoms for both substance P and bradykinin regardless of the precursor ion charge state. Reaction times less than 250 ms and efficiencies approaching 5% are compatible with on-line fragmentation, as would be desirable for bottom-up proteomics applications., (Copyright 2009 John Wiley & Sons, Ltd.)

The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The alpha(2B)-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates alpha(2B)-AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of alpha(2B)-AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of beta(2)-AR, alpha(1B)-AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.

This study offers experimental evidence for the existence of three pheromone races of the northern genitalic form of Phyllophaga anxia: one race in which females produce and males respond mainly to L-valine methyl ester, a second producing and responding to L-isoleucine methyl ester, and a third producing and responding to an intermediate range of blends of the two compounds. At Franklinville, NY, pheromone gland contents of females were analyzed using coupled gas chromatography-electroantennogram detection. Two types of females were found, one that produced greater than 99% L-valine methyl ester and another that produced greater than 99% L-isoleucine methyl ester. Capture-mark-release-recapture field tests with males at Franklinville established that most males were recaptured in traps baited with the same blends with which they were originally captured. The populations characterized at Franklinville, NY, have also been found at numerous locations from eastern Canada and the northeast and north central USA, sometimes in allopatry and sometimes in sympatry. At a site in Carver, MA, P. anxia males responded to blends of the methyl esters of L-valine and L-isoleucine, and Carver females produced blends similar to those to which the males responded. Populations responding to blends have been identified only from southeastern Massachusetts and Rhode Island. At a field site near Waterloo, NY, the addition of small proportions of L-isoleucine methyl ester to lures containing L-valine methyl ester did not affect trap captures, but higher proportions of L-isoleucine methyl ester were inhibitory, decreasing trap captures.

In peptide sequencing experiments involving a single step tandem mass acquisition, leucine and isoleucine are indistinguishable because both are characterized by a 113 Da mass difference from the other peptide fragments in the MS2 spectrum. In this work, we propose a new method to distinguish between these two amino acids in consecutive MSn experiments, exploiting a gas-phase fragmentation of isoleucine that leads to a diagnostic 69 Da ion. We used this method to assess the Leu/Ile residues of several synthetic peptides. The procedure was then tested on a tryptic digest of myoglobin, assigning the correct amino acid in the majority of the peptides. This work was performed with an old and low-resolution instrument, thus demonstrating that our method is suitable for a wide number of ion trap mass spectrometers, not necessarily expensive or up-to-date.

Apolipoprotein C-III (apoC-III) is an important regulator of lipoprotein metabolism. Radioisotope and stable isotope kinetic studies show differing results in relation to the kinetics of apoC-III in HDL. Kinetic analysis of HDL apoC-III may be difficult because of its low concentration, as well as the presence of other apoproteins at higher concentration, in the HDL fraction. We used Intralipid(R) (IL), known to preferentially extract apoC proteins from plasma, as a means of extracting apoC-III from HDL before apoprotein separation by isoelectric focusing gel electrophoresis for the measurement of tracer enrichment. Protein purity was assessed by an isoleucine-to-leucine (Ile/Leu) ratio, as apoC-III contains no isoleucine. We compared apoC-III kinetics in 14 men using a bolus infusion of deuterated leucine. The Ile/Leu ratio for IL-extracted HDL (IL-HDL) apoC-III (3.0 +/- 0.7%) was not different from that of VLDL apoC-III (2.6 +/- 0.6%) but was significantly lower than that of untreated HDL apoC-III (9.0 +/- 2.9%) (P < 0.001). The isotopic enrichment curves and fractional catabolic rates (FCRs) for IL-HDL apoC-III were not different from those of VLDL apoC-III. In contrast, HDL apoC-III had significantly lower isotopic enrichments and FCRs than IL-HDL apoC-III (P < 0.001). In conclusion, this simple IL method can be used to isolate apoC-III from HDL with minimal interference from other HDL apoproteins, and it demonstrates that the kinetics of apoC-III in VLDL and HDL are similar, supporting the concept of a single kinetically homogeneous pool of apoC-III in plasma.