Jerome M. Bailey

Chemical methods of protein sequence analysis

Chemical methods of protein sequence determination are reviewed with particular emphasis on methodology for increasing the sensitivity of amino-terminal sequence analysis and on progress toward the development of an automated procedure for sequential degradation from the carboxy-terminus.

Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: effects on catalysis, activation by sulfate, and thermal stability.

A “hinge-bending” domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Bankset al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183→Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. TheKm values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183→Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the Ala→Pro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183 → Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, withTm near 54°C, two transitions are evident for the mutant enzyme withTm values of about 45 and 54°C. Using a thermodynamic model for two interacting domains, a decrease in the free energy of domain-domain interactions of about 2 kcal was estimated from the DSC data.

Studies in C-terminal sequencing: New reagents for the synthesis of peptidylthiohydantoins

In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Baileyet al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50°C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.

An electrospray ionization study of some novel alkylamine thiohydantoin amino acid derivatives

An electrospray ionization-mass spectrometric (ES-MS) study of some novel alkylamine thiohydantoin amino acid derivatives is presented. The alkylamine derivatives are being developed as part of an on-going effort to couple an Edmanlike protein sequencer to a bench-top electrospray ionization (ES) mass spectrometer. The ES-MS and capillary-skimmer collision-induced dissociation (CID) mass spectra of eight dimethylaminopropylthiohydantoin (DMAP-TH) amino acid derivatives as well as the trimethylaminopropylthiohydantoin (TMAP-TH), diethylaminopropylthiohydantoin, and dibutylaminopropylthiohydantoin (DBAP-TH) derivatives of Phe are presented. The spectra contain prominent [M + H]+ ions as well as fragment ions due to the loss of the respective neutral alkylamines. The CID spectrum of DMAP-TH-Phe also contains the dibutylaminoethyl cation. The relative responses of the alkylamine thiohydantoin derivatives obtained under identical solvent conditions are found to increase as the solvophobicity of the amino acid R group increases; the most solvophobic DBAP-TH-Phe gives rise to the highest overall response. DMAP-TH-Phe and the quaternary amine derivative TMAP-TH-Phe have comparable sensitivities when a sufficiently acidic solvent (pH = 3) is employed. Implications of these studies in the coupling of a protein sequencer to an ES mass spectrometer are discussed as are the requisite modifications of a single quadrupole mass spectrometer for ES analyses.

C-terminal sequence analysis of polypeptides containing C-terminal proline

Publisher Summary The use of diphenylphosphoroisothiocyanatidate (DPP-ITC) and pyridine combines the activation and derivatization steps, and has permitted application of the C-terminal chemistry to a wide variety of protein samples with cycle times similar to those employed for N-terminal sequence analysis. This chapter describes the development of chemistry based on the DPP-ITC/pyridine reaction, which permits the efficient derivatization and hydrolysis of peptidyl C-terminal proline to a thiohydantoin and discusses the integration of this chemistry into an automated method for the C-terminal sequence analysis of polypeptides containing C-terminal proline. The chapter discusses automated chemistry, which is capable of the C-terminal sequence analysis of polypeptides containing Cterminal proline. The use of DPP-ITC/pyridine for derivatization permits the direct formation of an acylisothiocyanate at the C-terminus without the need for oxazolinone formation. Once an acylisothiocyanate is formed, it can cyclize to a quaternary amine, containing thiohydantoin. The quaternary amine, containing proline thiohydantoin can be readily cleaved with water vapor, or alternatively with the silanolate salt normally used for the cleavage reaction.

Automated C-Terminal Sequencing of Peptides and Proteins

The development of a chemical method for the sequential degradation of a protein or peptide from the carboxy-terminus is a goal of our laboratory. Such a method, in addition to complementing existing N-terminal methods of degradation, would be invaluable for the sequence analysis of proteins with naturally occurring N-terminal blocking groups and for the detection of post-translational processing at the carboxy-terminus of expressed gene products. Although several methods for a sequential C-terminal degradation have been proposed (Ward, 1986; Rangarajan, 1988), the thiocyanate method based on the procedure originally published by Schlack and Kumpf (1926) has been the most widely studied. Recent work in our laboratory, introducing new reagents for the derivatization of the C-terminal amino acid (Hawke et al., 1987) and for the specific cleavage of the derivatized amino acid (Bailey and Shively, 1991; Bailey et al., 1992), has suggested that an automated chemical method for the sequential degradation of polypeptides from the carboxy-terminus, analogous to the Edman method for amino terminal degradation, may be feasible.

Automated C-Terminal Sequencing of Polypeptides Containing C-Terminal Proline

There has been much interest in the development of a chemical method for the sequential C-terminal sequence analysis of proteins and peptides. Such a method would be analogous and complimentary to the Edman degradation commonly used for N-terminal sequence analysis (Edman, 1950). It would also be invaluable for the sequence analysis of proteins with naturally occurring N-terminal blocking groups, for the detection of post-translational processing at the carboxy-terminus of expressed gene products, and for assistance in the design of oligonucleotide probes for gene cloning. Although a number of methods have been described, the method known as the “thiocyanate method” (Schlack and Kumpf, 1926), has been the most widely studied and appears to offer the most promise due to its similarity to current methods of N-terminal sequence analysis.

Strategies For Increasing The Sensitivity of N-Terminal Sequence Analysis

Publisher Summary This chapter explores strategies for increasing the sensitivity of N-terminal sequence analysis. The kinetics of coupling with dimethylaminopropyl isothiocyanate (DMAP-NCS) is similar to those observed with phenyl isothiocyanate (PITC), with the exception that inclusion of an organic base is not necessary. The dimethylaminopropyl group of the reagent acts as its own base. In a study described in the chapter, the derivatization reaction to form the dimethylaminopropylthiourea derivative at the N-terminus of the pentapeptide was found to be complete within 10 min. Cleavage of this peptide with neat trifluoroacetic acid to generate a shortened N-1 peptide was found to be complete within 3 min at 50°C. Eight of the dimethylaminopropvl-thiohydantoin (DMAP-TH) standards have also been reacted with methyl iodide to synthesize the quaternary amine trimethylaminopropyl-TH analogues. This provides a means to compare the sensitivity of detection by electrospray mass spectrometry of the tertiary amine analogues that must act as proton acceptors to become positively charged versus the quaternary amine analogues, which carry a permanent positive charge.