scholarly journals Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Almut Hesse ◽  
Michael G. Weller
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Limit Of Detection ◽  
Certified Reference Materials ◽  
Internal Standard ◽  
Aromatic Amino Acids ◽  
Protein Quantification ◽  
Protein Determination ◽  
Relative Standard

Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports.

Amino Acid Analysis of Soybean Meal: Interlaboratory Study

1972 ◽  
Vol 55 (4) ◽  
pp. 686-691 ◽  
Author(s):  
J F Cavins ◽  
W F Kwolek ◽  
G E Inglett ◽  
J C Cowan
Keyword(s):  
Amino Acids ◽  
Statistical Analysis ◽  
Amino Acid ◽  
Standard Deviation ◽  
Relative Standard Deviation ◽  
Soybean Meal ◽  
Amino Acid Analysis ◽  
Mesh Size ◽  
Interlaboratory Study ◽  
Relative Standard

Abstract Amino acid analysis of soybean meal was studied by 5 laboratories. Between- and within-laboratory variations were significant for most amino acids, whereas variations due to hydrolysis procedure and sample mesh size in the under 30 to under 270 mesh range were significant at the 0.05 level for only 2 amino acids. The relative standard deviation was different for each amino acid with cystine, methionine, and ammonia having the highest values. Normalization of results to 95% nitrogen recovery had only a small effect on statistical analysis of the data. Values from special analytical procedures for cystine did not agree, whereas those for tryptophan agreed very well.

t-PA PROTEIN DETERMINATION: IMPROVED ACCURACY THROUGH COMPOSITION CONSIOUS AMINO ACID ANALYSIS (CCAAA)

1987 ◽  
Author(s):  
A Brändström ◽  
M Rånby
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Standard Deviation ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Amino Acid Analysis ◽  
Reference Method ◽  
Protein Determination ◽  
Molar Quantity ◽  
The Mean

Protein determination by composition consious amino acid analysis (CCAAA) is generally applicable to polypeptides with known amino acid composition.Traditionally, in protein determination by AAA the mass of protein substance in a preparation is quantitated by simple summation of the mass of all amino acid residues found, although some amino acids e.g. Trp, Ser, Thr, Cys are unsatisfactorily determined. In this study we have explored the, possibility of improving protein determination by AAA in cases where the amino acid composition of the preparation is precisely known from sequence data. In CCAAA protein determination the molar amounts of amino acids that are known to be quantitatively recovered are divided by the number of residues known to be present in the polypeptide. The mean of these values is identified as the molar quantity of polypeptide in the sample. In addition, the standard deviation of the mean serves as identification and homogeneity control. In this study the accuracy of the method was checked gravimetrically.CCAAA was applied on t-PA prepared from cultured Bowes melanoma cells by immunopurification on PAMl-Sepharose 6BFF and subsequent gelfiltration in 1 mol/L NH4HCO3 on Sephacryl SA-200. About 2 pg of lyophilized protein was hydrolysed in 6 mol/L HC1 under vacuum for 24 hours at 110°C and amino acid analysed. Amino acids selected for calculation of the molar quantity were (number of residues within parentheses) Asp+Asn(50), Glu+Gln(52), Pro(29), Ala(32.5), Val(25), Leu(39), His(16), Lys(21) and Arg(35.5). To obtain the gravimetric quantity the molar quantity was multiplied with the theoretical molecular weight as calculated from the sequence and with the carbohydrate content taken into account.For t-PA the standard deviation of the mean molar quantity typically was less than 5%. The result from CCAAA was about 10% higher than that obtained from conventional AAA. The result from CCAAA was within 10% of that found gravimetrically. The absorbtivity of t-PA was estimated to 1.75 (g/L)-1cm-1.In summary, composition consious amino acid analysis (CCAAA) is recommended as reference method for protein determination of well defined protein preparations where the amino acid composition is known.

Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

2018 ◽  
Author(s):  
Nidhi Gour ◽  
Bharti Koshti ◽  
Chandra Kanth P. ◽  
Dhruvi Shah ◽  
Vivek Shinh Kshatriya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Self Assembly ◽  
Aromatic Amino Acids ◽  
Neutral Condition ◽  
Single Amino Acid ◽  
Binding Assays ◽  
Human Neuroblastoma ◽  
Cytotoxicity Assays ◽  
First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

scholarly journals Determination of amino acids in human biological fluids by high-performance liquid chromatography: critical review

Amino Acids ◽  
2021 ◽  
Author(s):  
Grażyna Gałęzowska ◽  
Joanna Ratajczyk ◽  
Lidia Wolska
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Performance ◽  
Hplc Analysis ◽  
Limit Of Detection ◽  
Biological Fluids ◽  
Analytical Techniques ◽  
Epidemiological Studies ◽  
Preparation Conditions

AbstractThe quantitation and qualification of amino acids are most commonly used in clinical and epidemiological studies, and provide an excellent way of monitoring compounds in human fluids which have not been monitored previously, to prevent some diseases. Because of this, it is not surprising that scientific interest in evaluating these compounds has resurfaced in recent years and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC analytics on the basis of publications from the last few years. It helps to update and systematize knowledge in this area. Particular attention is paid to the progress of analytical methods, pointing out the advantages and drawbacks of the various techniques used for the preparation, separation and determination of amino acids. Depending on the type of sample, the preparation conditions for HPLC analysis change. For this reason, the review has focused on three types of samples, namely urine, blood and cerebrospinal fluid. Despite time-consuming sample preparation before HPLC analysis, an additional derivatization technique should be used, depending on the detection technique used. There are proposals for columns that are specially modified for amino acid separation without derivatization, but the limit of detection of the substance is less beneficial. In view of the fact that amino acid analyses have been performed for years and new solutions may generate increased costs, it may turn out that older proposals are much more advantageous.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals Involvement of calcium-sensing receptor activation in the alleviation of intestinal inflammation in a piglet model by dietary aromatic amino acid supplementation

2018 ◽  
Vol 120 (12) ◽  
pp. 1321-1331 ◽  
Author(s):  
Hongnan Liu ◽  
Bie Tan ◽  
Bo Huang ◽  
Jianjun Li ◽  
Jing Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Inflammatory Cytokines ◽  
Intestinal Inflammation ◽  
Aromatic Amino Acids ◽  
Dietary Supplementation ◽  
Basal Diet ◽  
Signalling Pathway ◽  
Protein Levels ◽  
Pro Inflammatory Cytokines

AbstractCa2+-sensing receptor (CaSR) represents a potential therapeutic target for inflammatory bowel diseases and strongly prefers aromatic amino acid ligands. We investigated the regulatory effects of dietary supplementation with aromatic amino acids – tryptophan, phenylalanine and tyrosine (TPT) – on the CaSR signalling pathway and intestinal inflammatory response. The in vivo study was conducted with weanling piglets using a 2 × 2 factorial arrangement in a randomised complete block design. Piglets were fed a basal diet or a basal diet supplemented with TPT and with or without inflammatory challenge. The in vitro study was performed in porcine intestinal epithelial cell line to investigate the effects of TPT on inflammatory response using NPS-2143 to inhibit CaSR. Dietary supplementation of TPT alleviated histopathological injury and decreased myeloperoxidase activity in intestine challenged with lipopolysaccharide. Dietary supplementation of TPT decreased serum concentration of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-12, granulocyte-macrophage colony-stimulating factor, TNF-α), as well as the mRNA abundances of pro-inflammatory cytokines in intestine but enhanced anti-inflammatory cytokines IL-4 and transforming growth factor-β mRNA levels compared with pigs fed control diet and infected by lipopolysaccharide. Supplementation of TPT increased CaSR and phospholipase Cβ2 protein levels, but decreased inhibitor of NF-κB kinase α/β and inhibitor of NF-κB (IκB) protein levels in the lipopolysaccharide-challenged piglets. When the CaSR signalling pathway was blocked by NPS-2143, supplementation of TPT decreased the CaSR protein level, but enhanced phosphorylated NF-κB and IκB levels in IPEC-J2 cells. To conclude, supplementation of aromatic amino acids alleviated intestinal inflammation as mediated through the CaSR signalling pathway.

scholarly journals Validation of a New Sensitive Method for the Detection and Quantification of R and S-Epimers of Ergot Alkaloids in Canadian Spring Wheat Utilizing Deuterated Lysergic Acid Diethylamide as an Internal Standard

Toxins ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 22
Author(s):  
Jensen Cherewyk ◽  
Taylor Grusie-Ogilvie ◽  
Barry Blakley ◽  
Ahmad Al-Dissi
Keyword(s):  
Spring Wheat ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Ergot Alkaloids ◽  
Internal Standard ◽  
Lysergic Acid ◽  
Lysergic Acid Diethylamide ◽  
Relative Standard ◽  
Validation Parameters ◽  
Sensitive Method

Ergot sclerotia effect cereal crops intended for consumption. Ergot alkaloids within ergot sclerotia are assessed to ensure contamination is below safety standards established for human and animal health. Ergot alkaloids exist in two configurations, the R and S-epimers. It is important to quantify both configurations. The objective of this study was to validate a new ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantification of six R and six S-epimers of ergot alkaloids in hard red spring wheat utilizing deuterated lysergic acid diethylamide (LSD-D3) as an internal standard. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effects, recovery and precision were investigated. For the 12 epimers analyzed, low LOD and LOQ values were observed, allowing for the sensitive detection of ergot epimers. Matrix effects ranged between 101–113% in a representative wheat matrix. Recovery was 68.3–119.1% with an inter-day precision of <24% relative standard deviation (RSD). The validation parameters conform with previous studies and exhibit differences between the R and S-epimers which has been rarely documented. This new sensitive method allows for the use of a new internal standard and can be incorporated and applied to research or diagnostic laboratories.

scholarly journals Комбинационное рассеяние света в хирально чистых и рацемической фазах поликристаллов триптофана и тирозина

2019 ◽  
Vol 127 (10) ◽  
pp. 541
Author(s):  
В.С. Горелик ◽  
М.Ф. Умаров ◽  
Ю.П. Войнов
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Raman Spectra ◽  
Spectral Range ◽  
Phase State ◽  
Source Parameters ◽  
Low Frequency ◽  
Aromatic Amino Acids ◽  
Chiral Phase ◽  
Left Handed

AbstractRaman spectra of tryptophan and tyrosine polycrystals have been analyzed in a wide spectral range by fiber-optic spectroscopy. The Raman spectra have been recorded with a BWS465-785H spectrometer in the spectral range of 0–2700 cm^–1 using a 785-nm cw laser as an excitation source. Parameters of the Raman spectra are compared for three crystalline phase modifications of aromatic amino acids: left-handed, right-handed, and racemic phase. The presence of strong Raman satellites, the characteristics of which change depending on the type of the chiral phase state of amino acid, is found in the low-frequency Raman spectra of tryptophan and tyrosine amino acid lattices. The results obtained can be used for monitoring the chiral purity of bioactive preparations containing amino acids.

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