No abstract

The sequence of human betaB1-crystallin cDNA encoded a protein of 251 amino acids in length. Mass spectrometric analysis of intact betaB1 from young human lens confirmed the deduced amino acid sequence. Lenses of human donors newborn to 27 years of age also contained partially degraded forms of betaB1 missing 15, 33, 34, 35, 36, 39, 40, and 41 amino acid residues from their N-terminal extensions. The similarity of the cleavage site between residues 15 and 16 in human betaB1 to the cleavage occurring in bovine betaB1 suggested that lenses of both species may contain a similar proteolytic activity. The remaining cleavage sites occurring in human betaB1 did not closely match those occurring in other species, possibly due to the widely divergent amino acid sequence of the N-terminal extension of betaB1 amoung species. Results from animal models suggest that cleavage of the N-terminal extension of betaB1-crystallin could enhance protein insolubilization and cataract in lens. However, the presence of partially degraded betaB1-crystallins in both water-soluble and water-insoluble fractions of lenses of young donors suggested that the rate that proteolyzed betaB1-crystallins become water-insoluble is relatively slow in humans.

A maize (Zea mays L.) small heat shock protein (HSP), HSP22, was previously shown to accumulate to high levels in mitochondria during heat stress. Here we have purified native HSP22 and resolved the protein into three peaks using reverse phase high performance liquid chromatography. Mass spectrometry (MS) of the first two peaks revealed the presence of two HSP22 forms in each peak which differed in mass by 80 daltons (Da), indicative of a monophosphorylation. Phosphorylation of HSP22 by [␥-32 P]ATP was also observed in mitochondria labeled in vitro, but not when purified native HSP22 was similarly used, demonstrating that HSP22 does not autophosphorylate, implicating a kinase involvement in vivo. Collisionally induced dissociation tandem MS (CID MS/MS) identified Ser59 as the phosphorylated residue. We have also observed forms of HSP22 that result from alternative intron splicing. The two HSP22 proteins in the first peak were ϳ57 Da larger than the two HSP22 proteins in the second peak. MS analysis revealed that the ؉57-Da forms have an additional Gly residue directly N-terminal of the expected Asp 84 , which had been converted to an Asn residue. These results are the first demonstrations of phosphorylation and alternative intron splicing of a plant small HSP.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography–mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

Deamidation evaluation and mitigation is an important aspect of therapeutic antibody developability assessment. We investigated the structure and function of the Asn-Gly deamidation in a human anti-CD52 IgG1 antibody light chain complementarity-determining region 1, and risk mitigation through protein engineering. Antigen binding affinity was found to decrease about 400-fold when Asn33 was replaced with an Asp residue to mimic the deamidation product, suggesting significant impacts on antibody function. Other variants made at Asn33 (N33H, N33Q, N33H, N33R) were also found to result in significant loss of antigen binding affinity. The co-crystal structure of the antigen-binding fragment bound to a CD52 peptide mimetic was solved at 2.2Å (PDB code 6OBD), which revealed that Asn33 directly interacts with the CD52 phosphate group via a hydrogen bond. Gly34, but sits away from the binding interface, rendering it more amendable to mutagenesis without affecting affinity. Saturation mutants at Gly34 were prepared and subjected to forced deamidation by incubation at elevated pH and temperature. Three mutants (G34R, G34K and G34Q) showed increased resistance to deamidation by LC-MS peptide mapping, while maintaining high binding affinity to CD52 antigen measured by Biacore. A complement -dependent cytotoxicity assay indicated that these mutants function by triggering antibody effector function. This study illustrates the importance of structure-based design and extensive mutagenesis to mitigate antibody developability issues.