Determination Of Amino Acids Research Articles

Threads for tear film collection and support in quantitative amino acid analysis.

The collection of tears for chemical composition analysis is complicated by both the difficulty in sampling the tear film and the relatively low microliter volumes available for analysis. The experiments in this study are focused on the demonstration of a method for determining amino acids from tear samples. Phenol red thread was used to absorptively collect tear fluid for qualitative and quantitative analyses of amino acids in basal, reflex, and emotional tears. The thread is also used as a support for sample preparation followed by elution with a buffer. The phenol red indicator on the thread turns from yellow to red with 15-s tear absorption and allows accurate volume measurement from 100nL to over 1μL. Derivatization of amino acids was performed directly on the thread with primary amine reactive fluorescamine for fluorescence detection. Analyte elution was performed via centrifugation with the thread in a pipet tip suspended in a centrifuge tube. Collected tear eluate was analyzed via capillary electrophoresis with LED-induced fluorescence. Glycine, glutamate, and aspartate were baseline resolved and used for method characterization. Recoveries were at 50% for a single derivation and elution step but average recoveries near 90% were found with two-step processing. Glutamate and aspartate are shown to be stable stored on thread for 3days. Basal, reflex, and emotional tears were analyzed from three subjects showing distinct amino acid profiles for each tear type. The demonstration of this method may facilitate the development of routine tear compositional analysis to assess ocular health. Graphical Abstract Schematic drawing of thread-based tear collection and quantitative analysis.

Running Induces Gut Injury but Does Not Modulate Postprandial Release of Dietary Protein Derived-amino acids

Resistance exercise has been shown to induce intestinal injury that can impair dietary protein digestion and absorption kinetics during recovery from exercise in young men. Although, endurance exercise can induce pronounced intestinal ischemia and gut injury, the effect of this exercise modality on the subsequent postprandial appearance of dietary protein-derived amino acids has yet to be examined. PURPOSE: We aimed to determine the effects of treadmill running on markers of intestinal injury and the subsequent release of dietary protein- derived leucine in circulation during recovery from exercise in trained athletes. METHODS: In crossover trials, six trained men (27±2 y; BMI= 24±0.9 kg/m2; VO2peak=62±4 ml/kg/min) received a primed continuous infusion of L-[1-13C]leucine and ingested 19 g of intrinsically L-[5,5,5-2H3]leucine labeled egg protein with 60 g carbohydrates at rest and after 1 h of treadmill running at 70%VO2peak. Blood samples were drawn every 0.5-1 h during the infusion protocols to determine amino acid concentrations, leucine enrichments, and intestinal fatty acid binding protein (I-FABP) as an estimate of small intestine injury. RESULTS: I-FABP increased throughout exercise with values peaking immediately after exercise (156% above baseline) but returning to baseline within 30 min of recovery. I-FABP was stable before and after protein ingestion in the resting condition (P>0.05). Postprandial release of dietary protein derived leucine did not differ between the rest and exercise conditions (P=0.33). CONCLUSION: Running induces small intestinal injury but does not affect splanchnic extraction of leucine from dietary protein during recovery from endurance exercise in trained athletes. These data suggest that endurance exercise-induced gut injury does not compromise protein delivery of a mixed meal during post-exercise recovery. Supported by the UIUC Center on Health, Aging, and Disability and the Faculty of Kinesiology and Physical Education Research Grants

Effect of Ultrasound on Alkali Extraction Protein from Rice Dreg Flour

AbstractIn this study, Counter‐current ultrasound‐assisted alkali was employed to extract rice dreg protein (RDP) from rice dreg flour (RDF). The results indicated that extraction yield of RDP reached a maximum value at 0.08 mol/L alkali concentrate. It was only 43.20% in absence of ultrasound, whereas it reached up to 88.44% under ultrasonic condition. Amino acid composition, scanning electron microscopy, atomic force microscopy and fourier transform infrared spectra were used to determine amino acid composition, morphology, aggregates and microstructure of RDP. Ultrasound treatment could result in improving of hydrophobic amino acids content and changing of RDF and RDP microstructure, showing enhancement in the porosity of RDF particle, variation of RDP particle size. Thus, the acceleration of protein extraction was related to the changes of RDF and RDP microstructure caused by ultrasound. Counter‐current ultrasound‐assisted alkali extraction, as a modified method, was proved to be helpful in industrial manufacture of RDP from RDF.Practical ApplicationsLow‐frequency high‐intensity ultrasound has been used in many aspects in laboratory, such as extraction of components, pretreatment. Counter‐current ultrasound‐assisted extraction was proved to be helpful in industrial manufacture of protein because it can dramatically improve extraction rate of rice dreg protein, and Counter‐current ultrasound method can achieve continuous production of industrialization.

Chemical composition, amino acid digestibility, and true metabolizable energy of cowpeas as affected by roasting and extrusion processing treatments using the cecectomized rooster assay

SUMMARY Nutritional differences due to variety, source, and effects of heat processing on cowpeas (Vigna unguiculata) were assessed. The precision-fed cecectomized rooster assay was used to determine amino acid digestibility and TME of cowpeas. Mozambican cowpea (nhemba) treatments included either raw or roasted, whereas those (black-eyed beans) sourced from the United States were raw, roasted, or extruded. Crude protein content on a DM basis was similar for raw nhemba (296 g/kg) in comparison to raw black-eyed beans (299 g/kg). Raw nhemba had slightly higher amino acid content (except tryptophan) than raw black-eyed beans. The level of trypsin inhibitors of nhemba was 3 times higher than black-eyed beans (6,700 vs 2,200 TIU/g). Heat treatment generally had no effect on amino acid concentration except for lysine, which was reduced in both beans by heat. Additionally, all heat treatments reduced the concentration of trypsin inhibitors to below 2,000 TIU/g. Amino acid digestibility for the nhemba cowpeas was lower than for the black-eyed cowpeas except for tryptophan. Roasting had no effect on amino acid digestibility in black-eyed beans, but increased digestibility of several amino acids in nhemba cowpeas, and the average increase was 3.4 percentage units. The TMEn of raw nhemba cowpeas was significantly increased (2,806 vs 3,347 kcal/kg DM) by roasting and the TMEn of raw black-eyed cowpeas was significantly increased by extrusion (3,164 vs 3,535 kcal/kilogram DM).

Relationship between ureB Sequence Diversity, Urease Activity and Genotypic Variations of Different Helicobacter pylori Strains in Patients with Gastric Disorders.

Association of the severity of Helicobacter pylori induced diseases with virulence entity of the colonized strains was proven in some studies. Urease has been demonstrated as a potent virulence factor for H. pylori. The main aim of this study was investigation of the relationships of ureB sequence diversity, urease activity and virulence genotypes of different H. pylori strains with histopathological changes of gastric tissue in infected patients suffering from different gastric disorders. Analysis of the virulence genotypes in the isolated strains indicated significant associations between the presence of severe active gastritis and cagA+ (P = 0.039) or cagA/iceA1 genotypes (P = 0.026), and intestinal metaplasia and vacA m1 (P = 0.008) or vacA s1/m2 (P = 0.001) genotypes. Our results showed a 2.4-fold increased risk of peptic ulcer (95% CI: 0.483-11.93), compared with gastritis, in the infected patients who had dupA positive strains; however this association was not statistically significant. The results of urease activity showed a significant mean difference between the isolated strains from patients with PUD and NUD (P = 0.034). This activity was relatively higher among patients with intestinal metaplasia. Also a significant associa-tion was found between the lack of cagA and increased urease activity among the isolated strains (P = 0.036). While the greatest sequencevariation of ureB was detected in a strain from a patient with intestinal metaplasia, the sole determined amino acid change in UreB sequence (Ala201Thr, 30%), showed no influence on urease activity. In conclusion, the supposed role of H. pylori urease to form peptic ulcer and advancing of intestinal metaplasia was postulated in this study. Higher urease activity in the colonizing H. pylori strains that present specific virulence factors was indicated as a risk factor for promotion of histopathological changes of gastric tissue that advance gastric malignancy.

Development of BAX 826, a Polysialylated Full-Length rFVIII with Significantly Improved PK Properties

Post-translational glycosylation determines the pharmacodynamic and pharmacokinetic properties of therapeutic proteins. While desialylation of FVIII/VWF (asialo-FVIII/VWF) has a significantly reduced half-life compared to normal FVIII/VWF complex (Sodetz et al 1977), we introduced additional sialic acids on recombinant (r) FVIII to obtain the opposite: prolonged survival of rFVIII in the circulation. Additional sialic acid residues were introduced to rFVIII by covalently binding polysialic acid (PSA) to full length (FL) rFVIII. The resulting drug candidate BAX 826, polysialylated human rFVIII, is manufactured from octocog alfa which is expressed in Chinese Hamster Ovary Cells by a plasma/albumin free cell culture method and is the active substance in Baxalta´s licensed product ADVATE.The manufacturing process for BAX 826 comprises several steps, starting with the bulk drug substance (BDS) of the ADVATE process which is subjected to polysialylation, i.e. covalent attachment of PSA of an average molecular weight of 20 kDa to rFVIII. Polysialylation is followed by a sequence of chromatographic purification steps and concentration of the conjugate by an ultra-/diafiltration step leading to the pre-formulated BDS. Final formulation of the BDS includes a filling and lyophilization step to obtain the final drug product. The process described is suited to manufacture BAX 826 in large scale and showed a good batch to batch consistency, ensuring an equivalent product quality for each batch.BAX 826 was extensively structurally and functionally characterized. The methods used included reducing and non-reducing peptide mapping to determine amino acid sequence and post translational modifications, qualitative analysis of modification sites, SDS-PAGE and Western blot analysis, assessment of three dimensional structure similarities of BAX 826 and rFVIII, ADVATE, by Fourier-transformed infrared spectroscopy (FTIR), dynamic light scattering (DLS) and circular dichroism (CD). Functional characterization was performed by assessment of the kinetics of the assembly and activity of FIXa-FVIII (tenase) complex, determination of the rate of activation and inactivation of BAX 826 by thrombin, determination of overall hemostatic potency by a thrombin generation assay, measurement of the rate of inactivation of untreated or thrombin-activated BAX 826 by activated protein C, measurement of kinetics of the binding of BAX 826 to von Willebrand factor (VWF), to phospholipids, and to low-density lipoprotein (LDL)-receptor-related protein 1 (LRP1) by surface plasmon resonance spectroscopy.While BAX 826 retained full hemostatic functionality of FVIII as a co-factor of the tenase complex, and can thus be considered a fully active FVIII molecule, it was found to have a reduced binding to VWF and to LRP1. Together with the conception that VWF as FVIII's chaperone dictates the maximally achievable terminal half-life extension for FVIII reduced binding to VWF and FVIII's major clearance receptor LRP1likely explains the prolonged pharmacokinetic (PK) properties when comparing BAX 826 with unmodified FL rFVIII in animal models. In summary, BAX 826, a polysialylated rFVIII derivative, can be manufactured reproducibly without relevant changes to the protein structure characteristic for a functional FVIII molecule with high specific activity. DisclosuresTurecek:Baxalta Innovations GmbH: Employment. Siekmann:Baxalta Innovations GmbH: Employment. Mitterer:Baxalta Innovations GmH: Employment. Graninger:Baxalta Innovations GmbH: Employment. Schrenk:Baxalta Innovations GmbH: Employment. Matthiessen:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment. Hoellriegl:Baxalta Innovations GmbH: Employment. Putz:Baxalta Innovations GmbH: Employment. Schwarz:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.

A sleep-inducing peptide from the venom of the Indian cone snail Conus araneosus

The marine snail Conus araneosus has unusual significance due to its confined distribution to coastal regions of southeast India and Sri Lanka. Due to its relative scarceness, this species has been poorly studied. In this work, we characterized the venom of C. araneosus to identify new venom peptides. We identified 14 novel compounds. We determined amino acid sequences from chemically-modified and unmodified crude venom using liquid chromatography-electrospray ionization mass spectrometry and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Ten sequences showed six Cys residues arranged in a pattern that is most commonly associated with the M-superfamily of conotoxins. Four other sequences had four Cys residues in a pattern that is most commonly associated with the T-superfamily of conotoxins. The post-translationally modified residue (pyroglutamate) was determined at the N-terminus of two sequences, ar3h and ar3i respectively. In addition, two sequences, ar3g and ar3h were C-terminally amidated. At a dose of 2 nmol, peptide ar3j elicited sleep when injected intraperitoneally into mice. To our knowledge, this is the first report of a peptide from a molluscivorous cone snail with sleep-inducing effects in mice. The novel peptides characterized herein extend the repertoire of unique peptides derived from cone snails and may add value to the therapeutic promise of conotoxins.

'Genetic Coding' Reconsidered: An Analysis of Actual Usage.

This article reconsiders the theoretical role of the genetic code. By drawing on published and unpublished sources from the 1950s, I analyse how the code metaphor was actually employed by the scientists who first promoted its use. The analysis shows that the term ‘code’ picked out mechanism sketches, consisting of more or less detailed descriptions of ordinary molecular components, processes, and structural properties of the mechanism of protein synthesis. The sketches provided how-possibly explanations for the ordering of amino acids by nucleic acids (the ‘coding problem’). I argue that employing the code metaphor was justified in virtue of its descriptive-denotational and explanatory roles, and because it highlighted a similarity with conventional codes that was particularly salient at the time. 1 Introduction2 Coding Schemes in the 1950s 2.1 The research problem: Determining amino acid sequences 2.2 The solution: Mapping schemes or ‘codes’ 3 The Code Metaphor Played Descriptive and Explanatory Roles4 The ness of Codes and the Expendability of the Code Metaphor5 The Role of Arbitrariness6 Conclusions

Site-directed mutagenesis of HgcA and HgcB reveals amino acid residues important for mercury methylation.

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative "cap helix" region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

Liquid Chromatography-High Resolution Mass Spectrometry for Peptide Drug Quality Control.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed using three peptide drugs: salmon calcitonin, bivalirudin, and exenatide as model systems to assess the suitability of this approach for monitoring peptide drug product quality. Calcitonin and its related impurities displayed linear responses over the range from 0.1 to 10μM (R (2) values for calcitonin salmon, Glu(14)-calcitonin, and acetyl-calcitonin were 0.995, 0.996, and 0.993, respectively). Intra-assay precision in terms of relative standard deviation (%RSD) was less than 10% at all tested concentrations. The accuracy of the method was greater than 85% as measured by spiking 0.1, 0.3, and 1% of Glu(14)-calcitonin and acetyl-calcitonin into a stock calcitonin solution. Limits of detection for calcitonin, Glu(14)-calcitonin, and acetyl-calcitonin were 0.02, 0.03, and 0.04μM, respectively, indicating that an impurity present at less than 0.1% (0.1μM) of the drug product API concentration (107μM) could be detected. Method validation studies analyzing bivalirudin and exenatide drug products exhibited similar results to calcitonin salmon in regard to high selectivity, sensitivity, precision, and linearity. Added benefits of using LC-HRMS-based methods are the ability to also determine amino acid composition, confirm peptide sequence, and quantify impurities, even when they are co-eluting, within a single experiment. LC-HRMS represents a promising approach for the quality control of peptides including the measurement of any peptide-related impurities. While the development work performed here is focus on peptide drug products, the principles could be adapted to peptide drug substance.

Elucidation of a Raft-Partitioning Motif in Transmembrane Proteins

Eukaryotic plasma membranes (PMs) are believed to possess lateral lipid domains termed lipid rafts, which form functional platforms for membrane sorting and cell signalling. The function of these domains depends on their selective recruitment of specific membrane proteins. However the underlying structural features governing protein partition into lipid rafts remain unknown. In live cells, the nanoscopic size and subsecond lifetime of rafts presents great difficulty for measuring their properties and composition. Intact PMs isolated as Giant Plasma Membrane Vesicles (GPMVs) phase separate into two microscopic, stable lipid domains, with one of these domains possessing greater lipid order, lower diffusivity and enriching for canonical raft components. This microscopic raft phase separation thus presents an optimal system for measuring protein partitioning between coexisting membrane domains. Using GPMVs, we demonstrate that a protein's transmembrane domain (TMD) is a central determinant of raft affinity. A major factor governing raft partition was the length of the TMD, with longer TMDs preferring the thicker raft domains. Further, by systematic mutation of the TMD of LAT (Linker for activation of T-cells), we were able to determine amino acid sequences which are necessary and sufficient for raft partitioning and which highly resemble the α-helix oligomerization motif GxxxG. This resemblance suggested that TMD oligomerization is the structural basis for the discovered raft-partitioning motif. We confirmed this hypothesis by measure sequence-dependent TMD oligomerization by fluorescence lifetime microscopy (FLIM) combined with Forster resonance energy transfer (FRET). Finally, we demonstrate the biological significance of our observations by showing that raft partitioning determines sorting between organellar membranes. For the entire panel of TMD-variants, we observed a strong quantitative relationship between raft association and sorting to the plasma membrane, with non-raft mutants being sorted into the lysosomes for degradation.

Determination of amino acids in jams, fruits and pharmaceutical preparations by gas chromatography using trifluoroacetylacetone and ethylchloroformate as derivatizing reagents

A gas chromatography (GC)-flame ionization detection procedure was used to determine amino acids in a pharmaceutical preparation (Aminess N tablets), jams, juices and a vegetable.

Protein Requirements: Are We Ready for New Recommendations?

In this issue of The Journal of Nutrition, 2 articles on protein requirements in women authored by the same research group are presented. The first deals with protein requirements during early and late gestation (1), and the second reports on requirements in women >65 y of age (2). Both studies conclude that the protein Estimated Average Requirements (EARs) and the RDAs are higher than the current FAO/WHO (3) and Institute of Medicine (4) recommendations (Table 1). Similar findings indicating higher protein requirements in octogenarian women (5), young men (6), and children (7) were published previously by the same research group (Table 1). The determination of protein requirements in humans is a challenging undertaking because of the limitations inherent to all the methods available. Nitrogen balance, considered by many as the ‘‘gold standard,’’ is not only tedious and time consuming but, even under extremely controlled conditions, fails to account for all of the nitrogen ingested (8). For this reason, alternative methods based on the oxidation of tracers have been proposed. One of these methods is indicator amino acid oxidation (IAAO), which has been used extensively to determine amino acid and protein requirements by the authors of the 2 articles published in this issue of The Journal of Nutrition. The main appeal of the IAAO technique is that it is minimally invasive and requires only a brief adaptation, thereby facilitating the determination of multiple intake points in the same individual. In addition, because of the short dietary interventions, it allows for the study of requirements in infants, children, and other vulnerable groups. However, there has been considerable debate on the theory behind the IAAO method and its application (9, 10). Therefore, the higher protein requirements that this method has yielded have been questioned. To their credit, the authors reanalyzed the existing nitrogen balance data and concluded that the RDA for adults should be 0.99 g protein/d or ;24% higher than the FAO/WHO recommendations (6). Although current recommendations (0.8 g protein/d) appear to be sufficient to maintain nitrogen balance in healthy people >65 y old (11), some studies suggest that a higher protein intake (1.0–1.2 g protein/d) could be beneficial in order to maintain muscle mass and function, among other physiologic endpoints (12). Likewise, higher protein intakes (1.2–1.4 g protein/d) are recommended for endurance athletes (13), although college students were able to maintain nitrogen balance for 2mo at protein intakes as low as 0.6 g/d (14). Thus, it seems that the protein requirements for ‘‘optimal’’ health and function are higher than the requirements to maintain nitrogen and protein balance. Regardless, these ‘‘optimal’’ and often elusive endpoints cannot be determined with short-term protocols, such as the IAAO technique. Protein malnutrition is not considered to be prevalent in the United States (15), although a small fraction of adolescent females and women >71 y old have usual protein intakes below the FAO/WHO RDA. However, a strikingly different picture emerges if RDAs estimated by using the IAAO technique are considered. For women aged >65 y the RDA estimated by Rafii et al. (2) exceeds the usual protein intake of ;74% of the U.S. population (15) (Table 1). Furthermore, the RDAs estimated by Stephens et al. (1) in early and late pregnancy exceed the protein intakes of 64% and >87%, respectively, of pregnant women from the same population (16). These new recommendations based on the IAAO technique carry serious policy implications and can affect programs such as the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) (17), the National School Lunch Program (18), and the Elderly Nutrition Program (19), which serve 8, 30, and 3 million people, respectively, at a cost >$16 billion/y. The consequences for food supplementation programs targeted toward vulnerable populations in developing countries would be substantially greater. For these reasons I would like to echo the concluding remarks of Tang et al. (5) regarding the IAAO technique in that ‘‘the limitations of this short-term, noninvasive method underscore the need for new research that uses alternative experimental designs and measuring physiologic, morphologic, and healthrelated outcomes.’’

Determination of Amino Acids in Plasma and Nutritional Supplements by Capillary Electrophoresis with Copper(II) Coordination

A capillary zone electrophoresis (CZE) method was developed using interactions between amino acids and copper(II) without derivatization. The optimized separation buffer included 10% isopropanol, 50 mmol L−1 CuSO4, 50 mmol L−1 acetic acid, and 50 mmol L−1 sodium acetate at pH = 3.98 at a detection wavelength of 233 nm. This method allowed baseline separation and determination of ten amino acids in less than 21 min. Quality parameters such as linearity (r > 0.99), precision (relative standard deviation <2.51%), limits of detection (0.05 µg mL−1), and recovery (82.25% to 117.6%) were measured. The separation of amino acids in high salt solutions was explored. The method was used to directly determine amino acids in human plasma and nutritional supplements.

Overview of peptide and protein analysis by mass spectrometry.

Mass spectrometry is an indispensable tool for peptide and protein analysis owing to its speed, sensitivity, and versatility. It can be used to determine amino acid sequences of peptides, and to characterize a wide variety of post-translational modifications such as phosphorylation and glycosylation. Mass spectrometry can also be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples, which makes it an extremely powerful tool for systems biology studies. The main goals of this unit are to familiarize peptide and protein chemists and biologists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide.

AFAL: a web service for profiling amino acids surrounding ligands in proteins.

With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein–ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand–protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5′-triphosphate.Electronic supplementary materialThe online version of this article (doi:10.1007/s10822-014-9783-6) contains supplementary material, which is available to authorized users.

Rapid determination of oligopeptides and amino acids in soybean protein hydrolysates using high-resolution mass spectrometry.

Soybean protein hydrolysates (SPHs), especially oligopeptides, have shown a variety of functional properties, including immunomodulatory and anti-oxidant effects. Soybean protein hydrolysate products have been used as functional ingredients in food, sports nutrition or clinical nutrition. However, the mixture is mostly undefined due to its complex nature, containing peptides and minor amino acids as well as small proteins. To develop a specific and efficient method for the identification and structural characterisation of oligopeptides in SPHs, and to determine free amino acids in SPHs in the same analytical run, for evaluation of the chemical profile of SPH products. Accurate mass spectrometry (MS) datasets of SPH samples were recorded on a high-performance liquid chromatography (HPLC) tandem high-resolution (HR) MS system. Potential oligopeptides were tentatively characterised based on their elemental compositions and ring double bond equivalent (RDBE) values, as well as HRMS/MS data. The analytical method to determine amino acids was evaluated in terms of linearity, precision, apparent recovery and limits of detection and quantitation. In total, 186 oligopeptides spanning the mass range of m/z 200-1500 and three major free amino acids could be determined in SPH samples in a single sample injection. Ninety-nine oligopeptides were tentatively characterised. The sensitive and specific instrumental performances also permitted the determination of 19 amino acids with a limit of quantitation of ≤ 0.1 μg/mL. The HPLC-HRMS technique has proven to be an advantageous tool for the rapid characterisation of oligopeptides and determination of amino acids in soybean protein hydrolysates.

S-acylation anchors remorin proteins to the plasma membrane but does not primarily determine their localization in membrane microdomains.

Remorins are well-established marker proteins for plasma membrane microdomains. They specifically localize to the inner membrane leaflet despite an overall hydrophilic amino acid composition. Here, we determined amino acids and post-translational lipidations that are required for membrane association of remorin proteins. We used a combination of cell biological and biochemical approaches to localize remorin proteins and truncated variants of those in living cells and determined S-acylation on defined residues in these proteins. S-acylation of cysteine residues in a C-terminal hydrophobic core contributes to membrane association of most remorin proteins. While S-acylation patterns differ between members of this multi-gene family, initial membrane association is mediated by protein-protein or protein-lipid interactions. However, S-acylation is not a key determinant for the localization of remorins in membrane microdomains. Although remorins bind via a conserved mechanism to the plasma membrane, other membrane-resident proteins may be involved in the recruitment of remorins into membrane domains. S-acylation probably occurs after an initial targeting of the proteins to the plasma membrane and locks remorins in this compartment. As S-acylation is a reversible post-translational modification, stimulus-dependent intracellular trafficking of these proteins can be envisioned.

Validation of a reverse-phase high-performance liquid chromatography method for the determination of amino acids in gelatins by application of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent

In-house method validation was conducted to determine amino acid composition in gelatin by a pre-column derivatization procedure with the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation for 18 amino acids in less than 40min; the overall detection and quantitation limit for amino acids fell into ranges of 5.68–12.48 and 36.0–39.0pmol/μl, respectively; the matrix effect was not observed, and the linearity range was 37.5–1000pmol/μl. The accuracy (precision and recovery) analyses of the method were conducted under repeatable conditions on different days in random order. Method precision revealed by HorRat values was significantly less than 2, except for histidine with a precision of 2.19, and the method recoveries had a range of 80–115% except for alanine which was recovered at 79.4%. The findings were reproducible and accurately defined, and the method was found to be suited to routine analysis of amino acid composition in gelatin-based ingredients.

Amino acids involved in lubiprostone activation of human ClC‐2 (896.11)

Objective. The study’s objective was to determine amino acids important for activation of human ClC‐2 (hClC‐2) by lubiprostone. NMR studies of ClC‐0 by Alioth et al (J Mol Biol 369:1163‐9, 2007) identified an α‐helical region that interacts with ClC‐0 CBS domains, important to channel regulation, which contains one of two PKA phosphorylation sites unique to hClC‐2 (RGET691).Methods. Whole cell patch clamp of wild type and mutant hClC‐2 expressed in EBNA293 cells were carried out as described (Cuppoletti et al, Cell Biochem Biophys 66: 53‐63, 2013). Wild type rabbit and mouse ClC‐2 were also studied.Results. Single amino acid mutagenesis of the PKA phosphorylation site RGET691‐&gt;A shifted the EC50 for lubiprostone stimulation of wild type hClC‐2 from 27.6 ± 4.5 nM (4) to 445.8 ± 2.1 nM (3). The triple mutant RGET691‐&gt;AGAA lost all lubiprostone stimulation. Both mutants were still stimulated by forskolin/IBMX. Both rabbit and mouse ClC‐2 were also stimulated by lubiprostone with EC50s of 350 ± 37.9 nM (3) and 64.3 ± 3.7 nM (4), respectively.Conclusions. RGET691 plays an essential role in lubiprostone stimulation of human ClC‐2. In light of NMR evidence that this region may interact with CBS domains and contains the RGET691 phosphorylation site in hClC‐2, binding of lubiprostone to this region in human, rabbit and mouse ClC‐2 may alter the interaction of this region with CBS domains, leading to direct modulation of ClC‐2.Grant Funding Source: Supported by Sucampo AG