A Simple Approach to Assign Disulfide Connectivity Using Extracted Ion Chromatograms of Electron Transfer Dissociation Spectra

Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of ETD facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds

Purification and Characterization of L,(L/D)-aminopeptidase from Guinea Pig Serum

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-Diso- Gln-meso-DAP(vNH2)-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (KM 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (KM \ubc 0.6mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin