Validation of peptide mapping with electrospray mass spectrometry for recombinant proteins of biopharmaceutical interest and its applications as an identity test and a characterization tool.

In this paper, the steps required to validate a liquid chromatography peptide mapping method with mass spectrometric detection (LC-MS) for use as an identity test and characterization tool are presented. All aspects of peptide mapping are evaluated and optimized, including protein sample preparation (protein reduction, alkylation and enzymatic digestion), high performance liquid chromatography (HPLC) separation of the resulting peptides, and the use of a mass spectrometric detection. In addition, the validation of a single quadruple MS detector is described and the implementation of on-line electrospray ionization MS (ESI-MS) as an adjunct detector to support the investigation of peak differences is presented. Applications of peptide mapping with tandem MS using an electrospray ion-trap instrument throughout the biopharmaceutical product development cycle are discussed, including assessing protein product heterogeneity derived from post-translational modifications (e.g. multiple N- or C-termini, deamidation, oxidation and glycosylation) and protein degradation.

Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions.

The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered [Baburina et al. (1994) Biochemistry 33, 5630-5635] and is located on the beta domain [Arjunan et al. (1996) J. Mol. Biol. 256, 590], while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains. The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors. Protein chemical methods, in conjunction with [1-14C] and [3-3H] analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14. However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme. Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol. Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes. Other potential nucleophilic sites for the inhibitor [C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site] were also substituted by a nonnucleophilic side chain. All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process. It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound [1-14C]-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site. Upon combining the tritiated inhibitor with [2-14C]-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein. It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol. Among other known activators, ketomalonate was found to react at C221 only. Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center. The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme [forming a Cys221S(-) + HHis92 ion pair; see Baburina et al. (1996) Biochemistry 35, 10249-10255, and Baburina et al. (1998) Biochemistry 37, 1235-1244]. Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone. All three compounds reduced the Hill coefficient and hence appear to react at C221. It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation.

Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio.

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

Purification of the gamma-aminobutyric acidA/benzodiazepine receptor complex by immunoaffinity chromatography.

The bovine gamma-aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62-3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7-1986/1. High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the immunoaffinity-purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7-1986/1 affinity-purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS-PAGE.

O'-(epoxyalkyl)tyrosines and (epoxyalkyl)phenylalanine as irreversible inactivators of serine proteases: synthesis and inhibition mechanism.

A series of O'-(epoxyalkyl)tyrosines and a carboxy terminal (epoxyalkyl)tyrosine and -phenylalanine were synthesized as potential serine protease inhibitors. N-Acetyl derivatives showed irreversible inactivation vis-a-vis subtilisin, while the N-benzoyl ones were specific toward chymotrypsin. The most potent inactivation of chymotrypsin was achieved by a O'-(3,4-epoxybutyl)-L-tyrosine derivative. The inactivation was shown to be stereospecific since a D derivative led to no irreversible inactivation. Placement of the epoxyalkyl group at the carboxy terminus led to potent rapid inactivation. Under these conditions some of the activity was later recovered. The two classes of inactivators (O'-epoxyalkyl and carboxy-epoxyalkyl) appear to operate by different mechanisms. Most importantly, it was found that irreversible inactivation by O'-(epoxyalkyl)-L-tyrosine only resulted if the carboxy terminus was a substrate (i.e. a compound with free carboxy terminus did not lead to inactivation). The ultimate activity kinetic assay (Daniels, S. B.; et al. J. Biol. Chem. 1983, 258, 15046-15053.) indicated that the epoxyalkyl group on the phenolic oxygen had an optimal length of four carbons with respect to the turnover ratio (the ratio of molecules undergoing turnover compared to those that inactivate the enzyme) for chymotrypsin. A different kinetic assay (Ashani, Y.; Wins, P.; Wilson, I. B. Biochim. Biophys. Acta 1972, 284, 427-434.) demonstrated that substratelike turnover was proceeding at considerably slower rates than for the corresponding true substrates and with rate-limiting deacylation of the acyl-enzyme. Amino acid analysis subsequent to acid hydrolysis demonstrated that Met had been selectively alkylated by the O'-(epoxyalkyl)tyrosine derivative. By contrast, alpha-chymotrypsin inactivated with N-benzoyl-L-Phe-2,3-epoxypropyl ester then subjected to amino acid analysis showed no change in the content of any amino acid that would serve as a potential nucleophile to the inhibitor. Yet, the L-Phe content increased, indicating that a covalent bond had been formed between the inhibitor and the enzyme. Either the bond between the inhibitor and the enzyme did not withstand the hydrolytic conditions and/or there was less than 10% decrease in the amino acids with nucleophilic side chains upon inactivation. Finally, two tripeptides containing O'-(epoxyalkyl)-L-tryosines were synthesized [N-(tert-butoxycarbonyl)-L-alanyl-L-alanyl-O'-(2,3-epoxypropyl)-L-tyrosi ne ethyl ester and N-(trifluoroacetyl)-L-valyl-O'-(2,3-epoxypropyl)-L-tyrosyl-L-valine methyl ester] as potential elastase inhibitors and were found to reversibly and competitively inhibit porcine pancreatic elastase.

Synthesis of the pro-peptide of subtilisin BPN'.

Subtilisin, a bacterial serine protease, is secreted as pre-pro-subtilisin. Previously, we demonstrated that the pro-peptide moiety of intact pro-subtilisin can guide the folding of inactive protein to active enzyme both in an intramolecular (6) and intermolecular manner (18). Herein is reported the total chemical synthesis of the pro-sequence (77 amino acids) of pre-pro-subtilisin BPN' carried out by solid phase methods. The structure was confirmed by both sequencing and amino acid analysis of the fragment peptides resulting from a V-8 protease digest. Preliminary studies indicate that the synthetic pro-peptide itself can renature denatured subtilisin BPN'. This study demonstrates a novel method for examining protein folding with the aid of exogenously added synthetic peptides.

Amino acid analysis on polyvinylidene difluoride membranes.

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.

Preparation and chromatographic use of 5'-fluorescent-labelled DNA probes.

A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'-terminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversed-phase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.

A membrane-associated folate binding protein (FBP) and a soluble FBP, which is released into the culture medium, have been purified from human KB cells using affinity chromatography. By NaDodSO4/PAGE, both proteins have an apparent Mr of approximately 42,000. However, in the presence of Triton X-100, the soluble FBP eluted from a Sephadex G-150 column with an apparent Mr of approximately 40,000 (similar to NaDodSO4/PAGE) but the membrane-associated FBP eluted with an apparent Mr of approximately 160,000, indicating that this species contains a hydrophobic domain that interacts with the detergent micelles. The amino acid compositions of both forms of FBP were similar, especially with respect to the apolar amino acids. In addition, the 18 amino acids at the amino termini of both proteins were identical. The membrane FBP, following delipidation with chloroform/methanol, contained 7.1 mol of fatty acid per mol of protein, of which 4.7 mol was amide-linked and 2.4 mol was ester-linked. The soluble FBP contained only 0.05 mol of fatty acid per mol of protein. These studies indicate that the membrane FBP of KB cells contains covalently bound fatty acids that may serve to anchor the protein in the cell membrane.

Gas-phase hydrolysis of proteins and peptides.

A simplified gas-phase hydrolysis procedure for proteins and peptides is described. The apparatus consists of a glass vacuum desiccator, a ceramic plate, and a Teflon ring. The method was shown to give reproducible compositions for hydrolysis of human serum albumin and microanalysis of alpha-melanocyte stimulating hormone including the quantitation of as little as one residue of tryptophan. It minimizes sample handling and allows for the simultaneous hydrolysis of a large number of samples.

Proton magnetic resonance studies of the states of ionization of histidines in native and modified subtilisins.

A technique was developed to exchange the backbone -N-H protons in D2O in the native subtilisins Carlsberg and BPN (Novo) that resulted in clearly resolved proton resonances in the aromatic region of the nuclear magnetic resonance spectrum. pH titration curves for four of the five histidine C2-H resonances in subtilisin Carlsberg and five of the six in subtilisin BPN between 7.5 and 8.8 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt provided microscopic pKa's between 6.3 and 7.2 for both sources of the enzyme at ambient (approximately 22 degrees C) probe temperature. A resonance that titrated with a pKapp of 7.35 +/- 0.05 was observed in the 1H spectra only of the diisopropylphosphoryl derivatives of the subtilisins from both sources. The 31P NMR pH titration of the same preparations under identical conditions of solvent (D2O) and temperature gave a pKapp = 7.40 +/- 0.05 of the single titratable resonance. Both observations must pertain to His-64 at the active center. A resonance smaller than the others and titrating with a pKapp of 7.2 could also be observed in the native enzymes. This resonance was assigned to the catalytic center histidine since its pK corresponded to that derived from kinetic studies. No major perturbations in the chemical shifts or the pK's derived from the pH dependence of the observed resonances were apparent in the presence of saturating concentrations of the two putative transition-state analogues phenylboronic acid and bis [3,5-(trifluoromethyl)phenyl]boronic acid and in monoisopropylphosphorylsubtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)