Peptide amino acid analysis

Maly et al. (1980) irradiated E. coli 50 S ribosomes and found that protein L4 was specifically crosslinked to the 23 S RNA. Again a 1 1 adduct was isolated, and after pepsin digestion, label was found in two peptides. Amino acid analysis showed that the peptides were similar and that a Tyr residue (residue 45 of L4) was altered. In one peptide the altered residue was N-terminal and in the second peptide the residue was the fifth and modified differently from that in the first peptide, in such a way that Edman degradation was blocked. 

Structure Determination of Peptides Amino Acid Analysis 1088... 

The amino add analysis of all peptide chains on the resins indicated a ratio of Pro Val 6.6 6.0 (calcd. 6 6). The peptides were then cleaved from the resin with 30% HBr in acetic acid and chromatogra phed on sephadex LH-20 in 0.001 M HCl. 335 mg dodecapeptide was isolated. Hydrolysis followed by quantitative amino acid analysis gave a ratio of Pro Val - 6.0 5.6 (calcd. 6 6). Cycll2ation in DMF with Woodward s reagent K (see scheme below) yielded after purification 138 mg of needles of the desired cyc-lododecapeptide with one equiv of acetic add. The compound yielded a yellow adduct with potassium picrate, and here an analytically more acceptable ratio Pro Val of 1.03 1.00 (calcd. 1 1) was found. The mass spectrum contained a molecular ion peak. No other spectral measurements (lack of ORD, NMR) have been reported. For a thirty-six step synthesis in which each step may cause side-reaaions the characterization of the final product should, of course, be more elaborate. 

Automated amino acid analysis of peptides containing asparagine (Asn) and glutamine (Gin) residues gives a peak corresponding to ammonia. Why ... 

Mabbott, G., 1990. Qualitative amino acid analysis of small peptides by GC/ S. Journal of Chemical Education 67 441—445. 

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

Because the amount of time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. Figure 26.3 shows the results of amino acid analysis of a standard equimolar mixture of 17 a-amino acids. Typically, amino acid analysis requires about 100 picomoles (2-3 /xg) of sample for a protein containing about 200 residues. 

To determine the structure of a peptide or protein, the identity and amount of each amino acid present is first found by amino acid analysis. The peptide is... 

Furthermore, the distribution of this peptide is compatible with PCP receptor densities, with highest concentrations of both ligand and receptor in areas that could be relevant to the psychotomimetic properties of PCP. Amino acid analysis of an aliquot of the purified fraction shows the peptide to contain at least 26 residues. Because the peptide is blocked at the N-terminus, enzyme fragments have been generated and purified over HPLC for sequence determination. 

Stone, K. L. and Williams, K. R., High-performance liquid chromatographic peptide mapping and amino acid analysis in the subnanomole range, /. Chromatogr., 359, 203, 1986. 

Peterson, J. A., Lorenz, L. J., Risley, D. S., and Sandmann, B. J., Amino acid analysis of peptides using HPLC with evaporative light scattering detection, /. Liq. Chromatogr. Related Technol., 22, 1009, 1999. 

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. 

Amino acid analysis remains a characterization technique undertaken in many laboratories, in particular if the product is a peptide or small polypeptide (molecular mass <10 kDa.). The... 

Silk fibroin contains no cystine and the content of lysine and histidine is also low (about 1% in total), but it does contain tyrosine phenolic (13%) and serine alcoholic (16%) sidechains. Since glycine accounts for 44% of the total aminoacid content, an N-terminal glycine residue is reasonably representative of most of the primary amino dyeing sites in silk fibres. Amino acid analysis of hydrolysed reactive-dyed silk indicates that the reaction between fibroin and reactive dyes takes place mainly at the e-amino group of lysine, the imino group of histidine and the N-terminal amino group of the peptide chain. In an alkaline medium, the hydroxy groups of tyrosine and serine also react [114]. 

Final products are cleaved with TFA-H20 (19 1) (1 mL/50mg of resin) at 25°C for 1 h. The filtrate from the cleavage reaction is collected, combined with TFA washes (1 mL/50mg of resin) of the cleaved peptide-resin, and dried. Cleavage yields (>85%) are calculated by amino acid analysis. 

The separation power of CIEF often generates a high number of peaks even when relatively pure samples are analyzed. As already discussed, one of the advantages of CIEF is its potential micropreparative capabilities. Capillary IEF allows the collection of fractions that can be further analyzed by other methods. Some of the most widely used characterization tools include MS, peptide mapping, and amino acid analysis. 

---