Protein sequencing peptide derivatization

GLC is an important adjunct to protein sequence determination. Automatic "sequenators" based upon the approach developed by Edman are available and have been described in detail by Niall (60). The Edman degradation, summarized in Equation 9.5, makes use of methyl or phenylisothiocyanate which reacts with the N-terminus of a peptide. Exposure of the isothiocyanate derivative of the protein to acid results in cleavage of the terminal amino acid as a thiaxolinones and exposure of the next amine group on the peptide. Thus, the process can be repetitively carried out, each amino acid removed from the peptide, in a sequential manner. Thiazolinones rearrange in acid medium to form thiohydantoin derivatives of amino acids, some of which may be directly gas chromatographed others must be derivatized typically as trimethylsilyl derivatives. 

The inability of C-terminal proline to be derivatized to a thiohydantoin has been a major impediment to the development of a routine method for the C-terminal sequence analysis of proteins and peptides. Since the method was first described in 1926 (2), the derivatization of C-terminal proline has been problematic. While over the years a few investigators have reported the derivatization of proline, either with the free amino acid or on a peptide, to a thiohydantoin (6-8), others have been unable to obtain any experimental evidence for the formation of a thiohydantoin derivative of proline (9-12). Recently, utilizing a procedure similar to that described by Kubo et al. (6), Inglis et al. (13) have described the successful synthesis of thiohydantoin proline from N-acetylproline. This was done by the one-step reaction of acetic anhydride, acetic acid, trifluoroacetic acid, and ammonium thiocyanate with N-acetyl proline. We have reproduced this synthesis and further developed it to a large scale synthesis of TH-Proline. 

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... 

Figure 2.5 Integrated analytical system. A nine-position microfabricated device was coupled to an ITMS instrument via a transfer capillary and a microESI ion source. The inner surface of the transfer capillary (15 cm long, 75 pm i.d., 150 pm o.d.) was derivatized with 3-aminopropylsilane. The etched channels were 30 pm deep and 72-73 pm wide. The diameter of the reservoirs was 1mm. The sample flow was controlled by an array of computer-controlled high-voltage relays which are also schematically represented. The software controlled the sample flow from the different reservoirs, the generation of MS spectra, the selection of potential peptides, the generation of MS-MS spectra and the matching of the MS-MS spectra against a protein sequence database. (Adapted with permission from Ref. 6).

Thermooptical detection has been combined with CE and used for protein and peptide Edman degradation sequencing detection, native protein detection, as well as the analysis of derivatized amino acid mixtures. " Frequency-doubled argon-ion lasers have been employed to supply an UV (257 nm) pump beam for the analysis of dansylated amino acids as well as the analysis of etopside and etopside phosphate in human blood plasma. In addition, two-color thermooptical absorbance detectors have been constructed, where each laser serves a dual role of pump and probe this system can be used to detect analytes that absorb in differing regions of the electromagnetic spectrum. 

Here, we will describe a range of applications of MALDI-MS, from the concepts of in-depth analysis of purified proteins to applications of MALDI-MS in a broader, proteomics-based research where proteins are identified, characterized, and quantified. In addihon, issues of sample preparation, protein characterization and identification strategies and bioinformatic tools for data interpretation wiU be discussed. The concepts of peptide fragmentation, sequencing and derivatization, analysis of post-translational modifications and the clinical apphcations of MALDI-MS are also briefly outlined. 

Amino acid analysis is important not only from a clinical aspect— amino acid metabolism disorders can be fatal if not diagnosed early—but also as the basis for protein and peptide sequencing. Due to the combination of the small amounts of material used in an actual analysis and the lack of chromophores on most amino acid residues, derivatization is common practice. Derivatization may be done prior to either separation or detection. 

PITC has been used extensively in the sequencing of peptides and proteins and reactions under alkaline conditions with both primary and secondary amino acids. The methods of sample preparation and derivatization follow a stringent procedure which involves many labour-intensive stages. However, the resulting phenylthio-carbamyl-amino acids (PTC-AA s) are very stable, and the timing of the derivatization step is not as critical as when using OPA. 

Fig. 2 Distributions of OPA-derivatized amino acids and peptides chromatographed by the automatic online OPA/2-mercaptoethanol system. A and B 50 pmol of tryptic peptide digest of proteins M and R, respectively. The peak marked with an asterisk is due to the derivatizing reagents. Column 5-/zm Resolve C, (15 cm X 3.9 mm). Emission at 425 nm and excitation at 338 nm. A comparison between the sequences of peptides M,5 and R,5 is also shown.

Li and Lloyd were among the first to report the use of immobilized proteins as a CSP in CEC [141]. They packed silica particles derivatized with ar acid glycoprotein (AGP) into CE capillaries. Chiral sites are present in the peptide sequence of AGP, as well as in the carbohydrate units present. The presence of the chiral centers sets up the same type of diastereomeric interactions as with CDs, as well as a host of other interactions such as hydrogen bonding. At the... 

The chemical scheme for C-terminal sequencing is shown in Figure 2. The first step involves treatment of the peptide or protein sample with diisopropylethylamine in order to convert the C-terminal carboxylic acid into a carboxylate salt. Derivatization of the C-terminal amino acid to a thiohydantoin is accomplished with diphenylisothiocyanatidate (liquid phase) and pyridine (gas phase). The peptide is then extensively washed with ethyl acetate and acetonitrile to remove reaction by-products. The peptide is then treated briefly with gas phase trifluoroacetic acid, followed by water vapor in case the C-terminal residue is a proline (this treatment has no effect on residues which are not proline). The derivatized amino acid is then specifically cleaved with sodium or potassium trimethylsilanolate to generate a shortened peptide or protein which is ready for continued sequencing. In the case of a C-terminal proline which was already removed by water vapor, the silanolate treatment merely converts the C-terminal carboxylic acid group on the shortened peptide to a carboxylate. The thiohydantoin amino acid is then quantitated and identified by reverse-phase HPLC. 

In order to test the specificity of the thiol labeling by MIANS and the general efficacy of this method to subsequently isolate and identify reactive thiols, the A-chain of ricin was used as a model protein. Although the A-chain contains two cysteine (Cys) residues, only the C-terminal Cys that forms the intermolecular disulfide linkage to the B-chain is accessible upon reduction of the native protein under the conditions employed (Montfort et al., 1987). If MIANS is indeed specific for reactive thiols and the anti-MIANS affinity column is effective, only MIANS-labeled peptides corresponding to the C-terminal sequence of ricin A-chain should be obtained (with a blank cycle at the Cys position upon automated Edman sequencing, due to its derivatization). 

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