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Amino Acid Analysis: Methods and Protocols | NHBS Academic & Professional Books

Sozialwissenschaften und Wirtschaft. Geisteswissenschaften, Kunst, Musik. Catherine Cooper Hrsg. Gesamtpreis inkl. MwSt :. Print-on-Demand: Lieferdauer ca. This book describes a variety of amino acid analysis techniques and how each technique can be used to answer specific biological questions.

Protocols used for LC-MS analysis

The first two chapters in Amino Acid Analysis Protocols introduce the concepts, basic theory, and practice of amino acid analysis. The following chapters give detailed instructions on various methods and their applications. As highlighted, there are many different approaches to amino acid ana- sis, but in all cases the results depend heavily on the quality of the sample. Therefore a new way to desalt samples prior to hydrolysis is covered as an introductory chapter Chapter 3 , and most authors have devoted a section to sample preparation, especially to the collection and storage of bodily fluids.

Some of the amino acid analysis methods described in this book are based on HPLC separation and analysis after precolumn derivatization. Chromatographic resolution of leucine Leu and isoleucine Ile , that are isobaric and challenging to separate, was used to benchmark the method.

Unlike reverse phase chromatography methods paired with modifiers [ 21 , 24 , 25 , 26 ], this approach can be adapted to existing liquid chromatography systems without requiring extensive instrument clean-up time. Eluted amino acids were ionized by an electrospray approach ESI and detected using a multiple reaction monitoring MRM method parameters described in Additional file 1 : Table S1.

Amino acids were monitored as positive ions except for the oxidized form of cysteine, cysteic acid, which was detected in negative ion mode. A serial dilution of an unlabeled amino acid standard mixture 0. Glycine, alanine and serine had higher LODs of 8. Despite the differences, the isotope dilution strategy produced reproducible results because the factors contributing to variability affected the amino acids in unlabeled samples as well as the internal standards. Protein hydrolysis efficiency and processing losses were assessed by quantifying a known amount of a protein standard.

A preliminary hydrolysis test was performed using seven different methods described in literature [ 41 , 42 , 43 , 44 , 45 , 46 , 47 ] based on a combination of reaction conditions, with agents to promote or diminish oxidative reactions that maximize sensitive amino acid detection Additional file 2 : Figure S1.

Three methods that gave maximal recovery and had low standard errors for most of the amino acids were further pursued. Detection of the most labile amino acids is presented in Additional file 2 : Figure S1. Aliquots of labeled amino acid standards were included with each hydrolysis to control for individual losses during the process. Each amino acid was quantified through the ratio of analyte peak area relative to the equivalent isotopically labeled 13 C, 15 N internal standard. Total protein concentration was calculated as the sum of individual amounts of amino acids.

Though accounted for through the isotopic dilution strategy, losses for individual amino acids were determined by comparing peak areas of isotopically labeled standards with or without prior hydrolysis Fig. Since the amino acid standards do not require hydrolysis the comparison indicates the extent of amino acid degradation during the process. When quantitative hydrolysis is desired [ 15 , 46 , 47 ] the time needed to break all peptidyl bonds can result in additional side chemical reactions that alter or degrade amino acids.

Single point calibrants that normalize for losses in signal intensity such as: norvaline, norleucine, alpha-amino butyric acid or sarcosine have distinct responses and therefore cannot account for the differences in specific amino acids [ 15 ]. Standard addition methods i. Quantitation of individual amino acids upon hydrolysis using three different methods. Comparison of standards with and without hydrolysis indicated significant degradation of a small number of amino acids including cysteine, methionine and tryptophan [ 41 , 42 , 43 , 44 , 45 ].

Asparagine and aspartate, glutamine and glutamate were analyzed together because asparagine and glutamine are deamidated during the hydrolysis process. Deamidation of asparagine and glutamine to the corresponding carboxylic acids does not significantly impact quantification of total protein because the molecular masses are near equivalent i.

Cysteine Cys , methionine Met and tryptophan Trp are susceptible to oxidative degradation during hydrolysis resulting in variable recovery. These three amino acids are important targets for bioengineering and crop improvement, due to their relative low content in soybean protein despite being a rich source of other essential amino acids [ 51 ]; therefore, quantification of the labile amino acids was scrutinized during the method development.

When protein was treated with H 2 O 2 prior to hydrolysis the stable oxidized forms were generated. When the protein quantity was calculated by the sum of all amino acid amounts, the results were variable and significantly underestimated the total protein level i. The low protein estimates from the AccQ-Tag method reflect losses that could not be accounted for during hydrolysis and processing. The AccQ-Tag method is typically used to establish relative amino acid composition and involves several preparations using different hydrolysis approaches in parallel for each sample to quantify the maximum number of amino acids.

Accounting for amino acid losses is not possible, therefore if one preparation is used to enhance throughput the approach will be more apt to underestimate total protein level. Alternatively, when assessing a standard or pure protein with known sequence, the measured concentration of all or a subset of amino acids relative to the number of residues can be plotted and the regressed line of best fit used to establish the protein level [ 55 ]. Since this latter approach relies on knowledge of the sequence of the protein s or mole fractions of the amino acids present in the sample, it is not applicable to complex protein mixtures as in most biological samples.

Individual amino acids concentrations y-axis as a function of hydrolysis technique and the number of residues present in BSA. The trendline represents the expected amounts of individual amino acids for protein recovered. Values that are above the trendline represent overestimation and below trendline represents underestimation of specific amino acids. Defatted soybean meal soy flour , a complex biological sample that is a common source for food and feed protein applications, was obtained as a reference material SRM from the National Institute of Standards and Technology NIST.

Absence of Trp indicated interference from carbohydrates during hydrolysis [ 42 , 52 ] which can be removed through prior processing [ 56 , 57 , 58 ]. All twenty amino acids were resolved and quantified using a commercially available mix of labeled amino acids resulting in quantitative determination of protein levels.

The method provides a robust and efficient strategy that could be extended to accommodate the detection of non-proteinogenic and modified amino acids [ 37 , 38 , 39 ] and can be used for semi-high throughput protein analysis without the need for derivatization after hydrolysis. A graphical summary of the method is presented in Fig. HILIC separates most amino acids such that potentially all overlapping isotopologues are resolved Fig. The inset figures indicate separation of amino acids with overlapping masses that can be resolved by HILIC and used to quantify isotopologues. All data generated or analyzed during this study are included in this published article and its supplementary information files.

Protein measurement with the Folin phenol reagent.

Amino Acid Analysis Protocols / Edition 1

J Biol Chem. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. Measurement of protein using bicinchoninic acid. High-precision continuous-flow isotope ratio mass spectrometry.

Mass Spectrom Rev.

Background

Gibson RB. The determination of nitrogen by the kjeldahl method. J Am Chem Soc. Jones DB. Factors for converting percentages of nitrogen in foods and feeds into percentages of proteins. US Department Agriculture, Circular Converting nitrogen into protein—beyond 6. Crit Rev Food Sci Nutr. Spectrophotometric and colorimetric determination of protein concentration. Curr Protoc Mol Biol. Quantitation of Protein. In: Burgess RR, editor. Methods in Enzymology, vol.

Deutscher MP: Academic Press; Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Kruger NJ. The bradford method for protein quantitation. In: Walker JM, editor. The Protein Protocols Handbook. Totowa, NJ: Humana Press; Amino acid analysis and protein database compositional search as a rapid and inexpensive method to identify proteins.

Column chromatography of amino acids with fluorescence detection. J Chromatogr A. Reverse-phase liquid chromatographic analysis of amino acids after reaction with o-phthalaldehyde. Amino acid analysis. Curr Protoc Protein Sci. Automatic recording apparatus for use in chromatography of amino acids. Anal Chem. Quantification of isotope label. In: Schwender J, editor. Plant Metabolic Networks. New York, NY: Springer; J Exp Bot. High-throughput analysis of amino acids in plant materials by single quadrupole mass spectrometry.

Plant Methods. Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry. Determination of underivatized amino acids in microsamples of a yeast nutritional supplement by LC-MS following microwave assisted acid hydrolysis.

Anal Methods. Plant J. J Chromatogr B. Alpert AJ. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. Buszewski B, Noga S. Anal Bioanal Chem. Identification and quantification of nucleosides and nucleobases in Geosaurus and Leech by hydrophilic-interaction chromatography. Hydrophilic interaction liquid chromatography of anthranilic acid-labelled oligosaccharides with a 4-aminobenzoic acid ethyl ester-labelled dextran hydrolysate internal standard.

Development and validation of an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry method for rapid quantification of free amino acids in human urine. Amino Acids. Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry. Amino acid analysis using chromatography—mass spectrometry: An inter platform comparison study. J Pharm Biomed Anal. A high-throughput method for the quantitative determination of free amino acids in saccharomyces Cerevisiae by hydrophilic interaction chromatography—tandem mass spectrometry.

Budding Yeast: A Laboratory Manual.

Quantification of a protein or peptide

Robust analysis of underivatized free amino acids in soil by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry. A hydrophilic interaction chromatography-tandem mass spectrometry method for amino acid profiling in mussels. Rapid determination of amino acids in fruits of Ziziphus jujubaby hydrophilic interaction ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry.

J Agric Food Chem. Hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry for highly rapid and sensitive analysis of underivatized amino acids in functional foods. Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography HILIC coupled with tandem mass-spectrometry. J Inherit Metab Dis.

Rapid Commun Mass Spectrom. Correction for amino acid loss during acid hydrolysis of a purified protein. Hydrochloric acid hydrolysis of proteins and determination of tryptophan by reversed-phase high-performance liquid chromatography. Determination of methionine sulfoxide in biological materials using HPLC and its degradability in the rumen of cattle.

Anim Feed Sci Technol.

Table of contents

Rayner CJ. Protein hydrolysis of animal feeds for amino acid content. Rutherfurd SM. Accurate determination of the amino acid content of selected feedstuffs.


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