Janne Crawford

The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two-dimensional gel proteome maps. In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins. Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation. The plasma membrane complement was resolved by two-dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectometry. In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P-type H(+)-ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane-span Mrh1p, the low affinity Fe(++) transporter Fet4p, the twelve-span Ptr2p, and the plasma membrane anchored casein kinase Yck2p. The four transmembrane-span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span. Thus, combining subcellular fractionation with adapted two-dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins.

The development of biologics necessitates reliable assays to characterize and control Host Cell Protein (HCP) impurities. Liquid Chromatography-Mass Spectrometry (LC-MS)-based HCP assays have emerged as a powerful orthogonal method to HCP ELISA, providing detailed information on individual HCPs. In response to a growing need, the U.S Pharmacopeia (USP) introduced General Chapter < 1132.1 > , which provides the best practices and outlines three quantitative LC-MS methods. This study explores the practical application and validation readiness of the following strategies: A - Relative to Product Protein, B - Relative to Spiked-in Protein, and C - Relative to Spiked-in Peptide. Two common HCPs-Clusterin and Lipoprotein Lipase-were quantified using LC-MS in a purified mAb drug substance spiked with a CHO cell culture harvest to simulate in-process HCP levels. All three methods were demonstrated in the same samples and dilutions, enabling direct comparison of the three methods from a single dataset. Method performance was assessed according to ICH Q2(R2) guidelines for analytical method validation, focusing on linearity, accuracy, precision, and specificity. Results include a comparative assessment and discussion of the advantages and disadvantages, and application of each aforementioned HCP quantification method. This study provides practical insights into the implementation of USP < 1132.1 > supporting the growing role of LC-MS in HCP analysis for biologics development.