Phenylthiohydantoins detection

Reaction with phenylisothiocyanate (PITC) in alkaline conditions produces stable phenylthiocarbamyl (PTC) adducts which can be detected either in the ultraviolet below 250 nm or electrochemically. However, this method involves a complex derivatization procedure and offers poorer sensitivity than the alternatives available for individual amino acids. It is useful, however, in conjunction with the automated analysis of peptides when single derivatized residues can be cleaved and analysed after conversion in acidic conditions to phenylthiohydantoins. 

Sequence analysis is one of the most useful tools for determining disulfide connectivities by detecting the phenylthiohydantoins (PTH) of either cystine or cysteine derivatives. Sequencing can be carried out on the intact peptide, although most frequently disulfide-bridged fragments are subjected to this type of analysis. 

The Edman degradation method for polypeptide sequence determination. The sequence is determined one amino acid at a time, starting from the amino-terminal end of the polypeptide. First the polypeptide is reacted with phenylisothiocyanate to form a polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino-terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected spectrophoto-metrically. The remaining intact polypeptide, shortened by one amino acid, is then ready for further cycles of this procedure. A more sensitive reagent, dimethylaminoazobenzene isothiocyanate, can be used in place of phenylisothiocyanate. The chemistry is the same. 

The sequencing of a peptide (26) uses the well-known Edman degradation (27) of peptides, as shown in Fig. 2.2. The reaction cycle consists of reaction with phenylisothiocyanate followed by treatment with anhydrous TFA to promote cycliza-tion of the intermediate thiourea. Rearrangement induced by treatment with aqueous TFA produces the phenylthiohydantoin (PTH) of the N-terminal amino acid 2.2 and the tmncated peptide 2.3. The sequence is repeated through n cycles until the whole peptide is degraded. The PTHs produced are detected by HPLC-UV and their retention times are compared to those of 20 standard PTHs, one for each natural amino acid. 

Phenylthiohydantoin derivatization offers a special value because it is actually performed during Edman degradation, the sequencing technique mostly used for the determination of the primary structure of proteins and peptides. PTH derivatives are separated in many different stationary phases, in either normal- or re-versed-phase mode and are mostly detected at 254 nm [8,9]. Using radiolabeled proteins, sequencing of proteins down to the 1-100-pmol range can be achieved. The formed derivatives are basic and thus interact strongly with base silica materials. RP separations are mostly carried out in acidic conditions with the addition of appropriate buffers (sodium acetate mostly, but... 

The most popular method is automated Edman chemistry (mn on a sequencer), a method which removes one amino acid at a time from the N-terminus, resulting in the sequential liberation of phenylthiohydantoin (PTH) amino acids, which are identified by on-line HPLC analysis. Edman sequanators are completely automated high-sensitivity instrument systems that can routinely detect and identify as little as 0.2 pmol to 1 pmol of amino acid in a given cycle and carry out more than 20 cycles with 1 to 5 pmol of protein. Most peptides of length 3-30 amino acids can be sequenced completely. Although amino... 

Waldron, K. C. and Dovichi, N. J., Sub-femtomole determination of phenylthiohydantoin-amino acids Capillary electrophoresis and thermooptic detection. Ana/. Chem., 64,1396,1992. 

A similar problem associated with synthetic peptides of chain length > 5 amino acid residues is the identification of side reactions. Those involve substitutions on imidazole-, phenol- and indole moieties of histidine, tyrosine, and tryptophane as well as conversions, transamidation, and cyclizations of aspartic and glutamic side chains. As long as structural variations are stable under the reaction conditions of an Edman degradation, they can be detected from the proper phenylthiohydantoines in combination with H-NMR- and mass spectrometry. Quantitative amino acid analyses of impure peptides after acidic total hydrolysis do not indicate those structural deviations between main product and contaminations. 

Because of this uncertainly, we try, first, to control the course of the synthesis as completely as possible during its processing. Second, we try to determine the quality of the synthetic end product on polymer by the aid of Edman degradation with quantitative exploitation of the phenylthiohydantoines obtained [161,164]. In this way contaminations of the product by false sequences can be detected in relative amounts of as small as 0.1% of the main chain. Generally we experienced purer peptides synthesized than liberated from polymer by any detachment reaction. This can be demonstrated qualitatively by the aid of thin layer chromatograms of the crude peptide products released from the support after the cleavage reaction and by end-group determinations before and after peptide detachment. 

The release of free [ P]phosphate at a particular cycle, which results from P elimination during cyclization, indicates the presence a P.Ser or P.Thr residue. P.Tyr is stable to cyclization and is released as the anilinothiazolinone derivative of P.Tyr. This can be converted to the phenylthiohydantoin (PTH) derivative of P.Tyr by incubation in 0.1 N HCl for 20 min at 80°C. Marker PTH-P.Tyr is readily synthesized by reacting P.Tyr with phenylisothiocyanate as described above in step 2, and can be detected as a dark spot when the TLC plate is examined under a hand-held UV light. In addition to the cycle at which free p P]phosphate or p P]PTH-P.Tyr is released, information on the sequence of the peptide may be obtained by the electrophoretic mobility shifts detected at each cycle. Thus, if a positive or negatively charged amino acid is removed at a cycle before the phosphoamino acid, there will be a corresponding shift in the mobility of the peptide. Remember that if the peptide contains a C-terminal lysine this will react with phenylisothiocyanate at cycle 1, which will cause the loss of a positive charge. It may be difficult to determine the position of a second, more C-terminal phosphorylated residue present in the same peptide. 

Hood, " which incorporates substantial modifications in the design of the commercial Beckman instrument coupled with the computer assisted identification of phenylthiohydantoin derivatives by reversed-phase HPLC. These methods have enabled HunkapUler and Hood to determine 24 residues of the mouse fibroblast interferon amino-acid sequence starting with only 0-6 fig of material. This remarkable achievement is now being succeeded by developments in the same laboratory. A machine of even greater sensitivity, based on coupling and cleavage in the gas phase, has been constructed. The sensitivity of the automatic amino-acid sequencer is apparently beginning to surpass most of the analytical techniques commonly used to detect proteins. 

Keller et al. (1984). separated eross-linking amino acids of elastin by two-dimensional silica gel TLC using butanol-acetic acid-water (4 1 1) and propa-nol-NH,-water (8 1 11). Schwartz (1984) detected as little as 60 pmol of histidine phenylthiohydantoin by UV (366 nm) irradiation on a fluorescein-containing silica gel plate after development with ethanol-acetic acid (7 3). Henderson et al. (1985) reported that two-dimensional TLC was useful for screening aspartyl-glycosaminuria in the urine of children by heating ninhydrin-stained plates at 120°C. 

Bhushan and Ali (1987) tested amino acid separations on silica gel layers impregnated with various metal salts. Bhushan and Reddy (1989) reported the separation of phenylthiohydantoin (PTH) amino acids on silica gel with new mobile phases. Laskar and Basak (1988) de.scribed a new ninhydrin-based procedure that produced different colors and good sensitivity for amino acid detection. Bhushan and Reddy (1987) reviewed the TLC of PTH amino acids. Gankina et al. (1989) described a unidimensional multistep silica gel HPTLC method for the separation and identification of PTH and dansylamino acids. Bhushan et al. (1987) developed numerous solvent systems for effective separations of 2,4-dini-trophenyl-(DNP) amino acids. Bhushan (1988) reviewed the TLC resolution of enantiomeric amino acids and their derivatives. Kuhn et al. (1989) reported the amino acid enantiomer separation by TLC on cellulose of d- and L-tryptophan and methyltryptophan. Guenther (1988) determined TLC-separated enantiomers by densitometry. 

For metabolomics studies using IM-MS, electrospray ionization (ESI) is commonly used and the quantitative analysis will be different than the Ni. With ESI, internal standards, standard addition, isotope dilution, or external calibration curves are common practices for relative quantitative analysis. An example of ESI-IM-MS quantitative analysis was demonstrated for 20 phenylthiohydantoin (PTH)-derivatized amino acids, the final products in the Edman sequencing process of peptides and proteins. Detection limits for these amino acid derivatives ranged from 1.04 to 3.52 ng (less than 17 pmol).<" ° Quantitative analysis has not yet been established with ESI-IM-MS for applications to metabolomics. 

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