The relatively rapid and extensive characterization of the amino acid sequence and site-specific carbohydrate structures of a recombinant, reshaped human monoclonal antibody directed against respiratory syncytial virus (RSHZ19) is presented. The integrated strategy used a combination of mass spectrometric and conventional methodologies. Liquid chromatography/electrospray mass spectrometry was used for peptide mapping and selective identification of glycopeptides, and Edman degradation and tandem mass spectrometry were used to define the sequences of selected peptides. Matrix-assisted laser desorption/ionization mass spectrometry provided the M(r) of the intact protein and was used to characterize endo- and exoglycosidase digests of isolated glycopeptides to identify the glycosylation-site peptide and define the structures of the carbohydrates at that site. These experiments verified 99.1% of the light- and 99.3% of the heavy-chain amino acid sequences. The N and C termini of both chains were confirmed, and the nature and extent of heterogeneity at the N and C termini of the heavy chain were determined. Oxidation of a specific methionine residue to the sulfoxide was demonstrated by sequencing the N-terminally blocked peptide by tandem MS. Carbohydrate was found exclusively at Asn296 of the heavy chain. There was no evidence for a nonglycosylated form of the molecule or for the presence of O-linked carbohydrate. The qualitative distribution of glycoforms at this site was determined by MS of the isolated, tryptic glycopeptide and compared with results obtained by high-performance anion exchange chromatography and high-resolution gel permeation chromatography of oligosaccharides released by hydrazinolysis. The sequence and linkage of individual glycan species were determined using matrix-assisted laser desorption/ionization MS to monitor the results of a series of controlled digestions with specific exoglycosidases. The set of glycoforms consists predominantly of biantennary, core fucosylated carbohydrates lacking sialic acid. The present study is one of the first to directly evaluate the quantitative as well as qualitative consistency of the MS methods with conventional methods for carbohydrate analysis.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

The original version of this Article omitted the following from the Acknowledgements: 'We thank Robert Kirkpatrick for implementing the high throughput protein design strategy that enabled screening and triage of essential A. baumannii targets, based on whole genome sequencing and annotation of BM4454 strain; and Stephanie Van Horn, Allan Kwan, Elizabeth Valoret for A. baumannii genome sequencing and annotation.' Also, the original version omitted an acknowledgement to Prof. Lydia Tabernero as one of our collaborators for supplying the purified proteins used in the Tuberculosis screen. This has been corrected in both the PDF and HTML versions of the Article.