Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE.

Fragmentation of monoclonal antibodies has been routinely observed in non-reducing SDS-PAGE, mainly due to disulfide-bond scrambling catalyzed by free sulfhydryl groups, resulting in a method induced artifact. To minimize this artifact, alkylating agents like iodoacetamide (IAM) and N-ethylmaleimide (NEM) were commonly included in SDS sample buffer to block free sulfhydryls. However, the selection of agents and the applied concentrations differ from study to study. In addition, there is no direct comparison of these agents thus far, resulting in difficulties in selecting the suitable agent. To address these questions, we have tested the activities of IAM and NEM in inhibiting the fragment-band artifact of IgG4 monoclonal antibodies. Our data suggest that the inhibition activity of both agents is concentration dependent. Interestingly, 5mM NEM can achieve the same inhibition effect as 40 mM IAM. In addition, NEM still retained strong activity after prolonged sample heating, whereas IAM lost most of its activity. Overall, NEM appears to have a better inhibition effect than IAM on all tested IgG4 proteins, either with SDS-PAGE or CE-SDS methods. These observations demonstrate that NEM has stronger fragmentation inhibition activity than IAM, and thus is a more suitable alkylating agent for both SDS-PAGE and CE-SDS method to reduce this fragmentation artifact.

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.

Characterization of S-thiolation on secreted proteins from E. coli by mass spectrometry.

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.

Robustness of the long-range structure in denatured staphylococcal nuclease to changes in amino acid sequence.

A nativelike low-resolution structure has been shown to persist in the Delta 131 Delta denatured fragment of staphylococcal nuclease, even in the presence of 8 M urea. In this report, the physical-chemical basis of this structure is addressed by monitoring changes in structure reflected in residual dipolar couplings and diffusion coefficients as a function of changes in amino acid sequence. Ten large hydrophobic residues, previously shown to play dominant roles in the stability of the native state, are replaced with polar residues of similar shape. Modest increases in the Stokes radius determined by NMR methods result from replacement of five isoleucine/valine residues with threonine, one leucine with glutamine, and oxidation of four methionines to the sulfoxides. Yet in the presence of all ten hydrophobic to polar substitutions and 8 M urea, the NMR signature of a native-like topology is still largely intact. In addition, removal of 30 residues from either the N-terminus (which deletes a three-strand beta meander) or C-terminus (a long extended segment and the final alpha helix) produces only very small changes in long-range structure. These data indicate that both the general shape of the denatured state and the angular relationships of individual bond angles to the axes describing the spatial distribution of the protein chain are insensitive to large changes in the amino acid sequence, a finding consistent with the conclusion that the long-range structure of denatured proteins is encoded primarily by local steric interactions between side chains and the polypeptide backbone.

Molecular alignment of denatured states of staphylococcal nuclease with strained polyacrylamide gels and surfactant liquid crystalline phases.

Residual dipolar couplings reflect the orientation of vectors between pairs of magnetic nuclei relative to a unique set of molecular axes. Thus, unlike NOEs and scalar couplings, dipolar couplings provide access to long-range structural information. A prerequisite for measurement of these NMR parameters is imposition of a weak net alignment, most simply by forcing the macromolecules to tumble in an asymmetric environment that restricts some orientations more than others. In this report, several denatured forms of staphylococcal nuclease are aligned by using compressed and stretched polyacrylamide gels, a nonionic type of lipid bilayer disk or bicelle, and a liquid crystalline phase formed by a cationic lipid. All three types of media can be used at high urea concentrations. While polyacrylamide gels and bicelles produce similar alignment tensors through steric interactions, a liquid crystalline phase of cetylpyridinium bromide aligns denatured nuclease along a different set of axes, presumably through electrostatic effects. The analysis of residual dipolar couplings collected with two different alignment tensors may permit the calculation of ensembles of conformations. The dipolar couplings observed for staphylococcal nuclease denatured with urea, by low pH or by deletion of residues from both termini, suggest that all denatured forms share a common "topology", one which has been shown previously to be native-like. Although SDS/nuclease complexes give sharp and disperse (1)H-(15)N correlation spectra, only small couplings are observed in strained polyacrylamide gels.