Amino acid composition analysis

FIGURE 5.5 (a) The hydroxy amino acids serine and threonine are slowly destroyed during the course of protein hydrolysis for amino acid composition analysis. Extrapolation of the data back to time zero allows an accurate estimation of the amonnt of these amino acids originally present in the protein sample, (b) Peptide bonds involving hydrophobic amino acid residues snch as valine and isolencine resist hydrolysis by HCl. With time, these amino acids are released and their free concentrations approach a limiting value that can be approximated with reliability. 

After device construction, structural and functional analysis are critical. One might argue that only the second issue matters, but structural data often give insights into why devices perform suboptimally, and provide important clues about how to improve device function. We routinely use protein analytics (matrix-assisted laser desorption-ionization mass spectroscopy, amino acid composition analysis, gel electrophoresis, Western blotting, circular dichroism, vari-... 

An excellent review on protein hydrolysis for amino acid composition analysis has been published by Eountoulakis and Lahm [190], Hydrolysis can be performed by either chemical (under either acidic or basic conditions) or enzymatic means. The acidic hydrolysis itself can be carried out in a liquid or a gas-phase mode. The conventional acid hydrolysis uses 6M HCl for 20-24 h at 110°C under vacuum [200], In these conditions, asparagine and glutamine are completely hydrolyzed to aspartic acid and glutamic acid, respectively. Tryptophan is completely destroyed (particularly in the presence of high concentrations of carbohydrate), while cysteine and sometimes methionine are partially oxidized. Tyrosine, serine, and threonine are partially destroyed or hydrolyzed and correction factors have to be applied for precise quantification [190,201],... 

You have isolated a tetrameric NAD+-dependent dehydrogenase. You incubate this enzyme with iodo-acetamide in the absence or presence of NADH (at 10 times the Km concentration), and you periodically remove aliquots of the enzyme for activity measurements and amino acid composition analysis. The results of the analyses are shown in the table. 

M Fountoulakis, J-F Juranville, P Berndt. Large-scale identification of proteins of Haemophilus influenzae by amino acid composition analysis. Electrophoresis 18 ... 

The final product is controlled by electrospray ionization mass spectroscopy. If the mass obtained does not correspond to that predicted for the chemokine with the formation of two disulfide bonds, amino terminal sequencing and amino acid composition analysis is used to identify possible mutations. 

The number of methionine residues substituted by norleucine for isoforms F, G, and H was further confirmed by amino acid composition analysis. Each of the isolated Met->Nle isoforms was shown to have a composition consistent with the incorporation of one norleucine residue for one methionine residue. While no norleucine was found in the native r-metHuLeptin, [15N]r-metHuLeptin contains approximately 5% of norleucine relative to methionine. 

M. Fountoulakis and H.-W. Lahm, Hydrolysis and amino acid composition analysis of proteins, /. Chromatogr. A 826 109 (1998). 

The overall amino acid composition is determined using various hydrolytic and analytical procedures and compared with the amino acid composition deduced from the gene sequence for the desired product, or the natural counterpart, if considered necessary. In many cases, amino acid composition analysis provides some useful structural information for peptides and small proteins, but such data are generally less definitive for large proteins. Quantitative amino acid analysis data can also be used to determine protein content in many cases. 

In many cases, it will be desirable to determine the extinction coefficient (or molar absorptivity) for the desired product at a particular UV/VIS wavelength (e.g., 280 nm). The extinction coefficient is determined using UV/VIS spectrophotometry on a solution of the product having a known protein content as determined by techniques, such as amino acid compositional analysis or nitrogen determination. If UV absorption is used to measure protein content, the extinction coefficient for the particular product should be used. 

Various methods are available to determine such protein properties, including amino acid composition analysis [34, 35, 36, 37], partial Edman sequencing [38], peptide mass fingerprinting and MS/MS sequencing (for... 

The 2D gel or Iso-Dalt is the most commonly used method in proteomics because of its relatively easy use, automation, high reproducibility, high resolution of proteins, and applicability to analysis by mass spectroscopy. Furthermore, protein bands separated by Iso-Dalt are readily amenable to Edman degradation or to the amino acid composition analysis. 

Wheeler, C. H., S. L. Berry, M. R. Wilkins, J. M. Corbett, K. Ou, A. A. Gooley, 1. Humphery-Smith, K. L. WUhams, and M. J. Dunn. 1996, Characterisation of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid compositional analysis. Electrophoresis 17 580-587. 

A highly thermostable protein disulfide oxidoreductase was first isolated from Sulfolobus solfataricus. From its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol (DTT), the protein was considered a thioredoxin. The protein showed an unusually high molecular mass of 25 kDa and from amino acid composition analysis contained four cysteine residues. A homologous protein was subsequently purified from Pyrococcus furiosus. From its amino acid sequence, which showed two distinct CXXC motifs, and from its thioltransferase activity the protein was considered to be a glutaredoxin-like protein. 

The Pj-22 isoforms were further purified by reverse phase HPLC (separation on the basis of hydrophobicity data not shown) and subjected to amino acid composition analysis. The data are summarized in Table 1. Knowing the relative amounts of each amino acid will define an enzymatic fragmentation strategy that will permit determination of the total Pj-22 sequence. 

To overcome the low throughput characteristic of Edman degradation, amino acid composition analysis was implemented in the protein identification scheme. This method is based on the chromatographic analysis of free amino acids obtained after acid hydrolysis [13, 14, 15]. Amino acid analysis can achieve high throughput protein identification, however, there is a decrease in the confidence of identification [16, 17, 18]. A combination of amino acid analysis and Edman degradation, limited to 3-5 cycles to obtain a short sequence tag, was used to increase the confidence in protein identification [19, 20, 21]. 

Traditionally, proteins were initially characterized by de-novo sequencing using automated Edman degradation and amino acid composition analysis. Today, however, these techniques tend to be replaced by MS, which not only provides more flexibility and sensitivity but is also amenable to the analysis of protein and peptide mixtures. Tandem mass spectrometry (MS/MS) is used for amino acid sequencing of peptides. MALDI-MS/MS is very powerful for peptide characterization and identiflcation via sequencing and sequence database searching. 

Schegg, K.M. Denslow, N.D. Andersen, T.T. Bao, Y. Cohen, S.A. Mahrenholz, A.M. Mann, K. Quantitation and identification of proteins by amino acid analysis ABRF-96AAA collaborative trial. In Techniques in Protein Chemistry, 8th Ed. Marshak, D.R., Ed. Academic Press San Diego, CA, 1997, 207-216. Fountoulakis, M. Lahm, H.-W. Hydrolysis and amino acid composition analysis of proteins. J. Chromatogr. A, 1998, 826, 109-134. 

For routine determination of unknown N-terminal protein sequences, at least 50-100 pmole of the purified protein should be available. The only method for the accurate quantitation of low picomole amounts of protein is quantitative amino acid composition analysis. Twenty to fifty picomoles of protein should be set aside... 

From this solution remove a 5-10% aliquot for quantitation by amino acid composition analysis. 

Figure 13 Comparative peptide mapping of two forms of rSmp28 by LC-MS (postcolumn stream splitting see Fig. 3). (A) Analysis of the nonmodified and (B) of the modified protein ( labels the nonmodified and O the modified N-terminal peptide). The on-line mass spectra of the nonmodified (C) and the modified (D) N-terminal peptides indicate a difference of 26.1 Da, and amino acid composition analysis of the collected peptide fractions gave identical results (expected sequence AGEHIK). The modified peptide was inaccessible to Edman degradation, lacked two deuterium-exchangeable hydrogen atoms, and could be separated into two isomers. Detailed analysis by tandem mass spectrometry showed that it was modified due to addition of acetaldehyde to a H, and K (see Fig. 14 and Ref. 44). (From Ref. 44.)...

The majority of amine-initiated block copolypeptides were often subjected to only limited characterization (e.g., amino acid compositional analysis) and, as such, their structures, and the presence of homopolymer contaminants, were not conclusively determined. Some copolymers, which had been subjected to chromatography, showed polymodal molecular weight distributions containing substantial high- and low-molecular-weight fractions.The compositions of these copolymers were found to be different from the initial monomer feed compositions and varied widely for different molecular weight fractions. It appears that most, if not all, block copolypeptides prepared using amine initiators have structures different than predicted by monomer feed compositions and likely have considerable homopolymer contamination due to the side reactions described above. 

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