Protein phenylthiohydantoin derivative

Automated Edman degradation of protein and peptide samples were performed using an Applied Biosystems sequencer (Model 470A or 477A) or a Hewlett Packard G1005A sequencer. Each sequencer was fitted with an on-line HPLC analyzer for the identification of phenylthiohydantoin derivatives. 

The TLC method was developed for amino acids therefore, in principle, it is equally applicable to peptides. Peptides, such as amino acids, are generally hydrophilic. There are, however, limits to this analogy. Dinitrophenylamino acid derivatives (DNP amino acids) and phenylthiohydantoin derivatives (PTH amino acids) are obtained when reaction of peptides or proteins with dinitrofluorobenzene or phenyl mustards are properly degraded. Their separation from reaction mixtures and their identification are considerable practical importance because they constitute essential steps... 

Acetonitrile has found wide use in the separation of amino acids, peptides and proteins. A mainstay separation is that of derivatized amino acids. Classic precolumn derivatization methods include phenyl isothiocyanate (PITC) to give the PTH (phenylthiohydantoin) derivative, dimethylaminonapthalenesulfonyl (dansyl) chloride, o-phthalaldehyde (OPA) and 9-fluoromethylchloroformate (FMOC). From there, many variations on a theme have been developed. (The reader is referred to Chapter 4 for some of them.)... 

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. 

First, the peptide is converted to its N-terminal PCT derivative by treatment with phenyhsothiocyanate. Next, the PCT protein is treated with trifluoroacetic acid, then with water to give the phenylthiohydantoin derivative. The N-terminal amino acid is released in this step. The other peptide bonds are not affected. 

Edman degradation (Section 27 13) Method for determining the N terminal amino acid of a peptide or protein It in volves treating the material with phenyl isothiocyanate (CgH5N=C=S) cleaving with acid and then identifying the phenylthiohydantoin (PTH derivative) produced Elastomer (Section 10 11) A synthetic polymer that possesses elasticity... 

Sun, T., and R.E. Lovins, Quantitative protein sequencing using mass spectrometry use of low ionizing voltages in mass spectral analysis of methyl- and phenylthiohydantoin amino acid derivatives. Anal Biochem, 1972. 45(1) 176-91. 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

The structure of peptides containing 20 eukaryotic natural amino acids is now routinely determined by the use of automatic protein microsequencer, which uses Edman chemistry to convert each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivative. The formed PTH-amino acids can be identified by their retention times on HPLC systems by comparison with reference standards derived from the 20 natural amino acids. For an OBOC peptide library composed of natural amino acids, the sequencing protocols of the automatic sequencer are well developed and standardized. However, structure determination of peptides composed of unnatural a-amino acids requires modification of the standard sequencing program.32 For peptides composed of non-a-amino acids, one can use an encoding strategy or mass spectrometry if a cleavable linker is employed. In this chapter, we shall focus on the new sequencing method we have developed for unnatural a-amino acids. 

The primary structure (i.e., the amino acid sequence) of a protein can be determined by stepwise chemical degradation of the purified protein. By far the most powerful and commonly used technique for doing this is the automated Edman degradation. The amino terminal amino acid residue of the polypeptide is reacted with Edman s reagent (phenylisothiocyanate) to form the phenylthiocar-bamyl derivative, which is removed without hydrolysis of the other peptide bonds by cyclization in anhydrous acid. The amino acid derivative is converted to the more stable phenylthiohydantoin and identified by HPLC. The process can be repeated many times, removing the amino acids from the amino terminus of the polypeptide one residue at a time and identifying them until the entire sequence... 

Conversion of half-cystine residues in proteins and peptides to the S-methyl derivatives is advantageous in subsequent studies of amino acid sequence. Under the usual conditions of acid hydrolysis ( 2.1), S-methylcysteine is recovered in a 90% yield (Heinrikson 1971). The phenylthiohydantoin of S-methylcysteine is readily identified by routine thin layer chromatography procedures (Rochat et al. 1970). With the increasing use of the sequenator, PTH-S-methylcysteine offers a marked advantage over derivatives such as PTH-cysteic acid, or PTFl-carboxymethylcysteine, which have to be identified by special techniques (Edman 1960, 1970). S-methylcysteinyl residues provide a new point of cleavage for cyanogen bromide (5). 

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... 

H- and 13C-NMR data have been reported for diagnostic purposes in direct analysis of phenylthiohydantoin amino acid derivatives (PTH) produced in the Edman degradation of peptides and proteins.189-193 The insensitivity of 3H-NMR spectroscopy constitutes a major hurdle for its application in the sequence study of peptides.194,195 Alternatively, identification of the cleaved amino acids in the automated Edman degradation has been solved in some cases by using IR,196-198 mass,199 and gas chromatographic techniques.200... 

There are several ways to identify the N-terminal amino acid of a peptide or protein. One of the most widely used methods is to treat the protein with phenyl isothiocyanate (PITC), more commonly known as Edman s reagent. This reagent reacts with the N-terminal amino group, and the resulting thiazolinone derivative is cleaved from the protein under mildly acidic conditions. The thiazolinone derivative is extracted into an organic solvent and in the presence of acid, rearranges to a more stable phenylthiohydantoin (PTH). 

The sequencing methods and determination of C-terminal and N-terminal amino acids are now widely used in biochemical research. The identification and quantitation of the characteristic degradation products can be accomplished by the gas-phase analytical methods. Thus, GC of both dinitrophenyl and various hydantoin amino acid derivatives has now been widely documented. Separation of thiohydantoins [244,245], phenylthiohydantoins [490,491] and methylthiohydantoins [492] generally requires additional silylation for the sake of volatility. Furthermore, acyl derivatives of similar substances have also been reported [493,494]. The most obvious advantage of GC for determination of the Edman degradation products is sensitivity which is particularly important in the sequence analysis of only minute amounts of proteins and peptide hormones. 

Determining the structure of a peptide or protein is carried out in several steps. The identity and amount of each amino acid present in a peptide is determined by amino acid analysis. The peptide is hydrolyzed to its constituent a-amino adds, which are then separated and identified. Next, the peptide is sequenced. Edman degradation by treatment with phenyl isothiocyanate (PITC) cleaves one residue from the N terminus of the peptide and forms an easily identifiable phenylthiohydantoin (PTH) derivative of the N-tenninal amino acid. A series of sequential Edman degradations allows the sequendng of a peptide chain up to 50 residues in length. 

Separation of phenylthiohydantoin (PTH) amino acid derivatives, which are produced in the sequential analysis of proteins by the Edman degradation, is required. Ashraf-Khorassani etal. showed [21] how SFC on a cyanopropyl-modified silica column allowed the separation of more than twenty PTH-amino acids if an ion-pairing reagent (tetramethylammonium hydroxide) was present at low concentration in the methanol-modified CO2 mobile phase. 

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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