Mass spectrometry is a sensitive and specific analytical technique that is capable of providing qualitative and quantitative data to resolve the protein elements of biochemical pathways that are altered by antibiotics. Here we present methods to study antibiotic susceptibility by changes in protein abundance, as exemplified by Klebsiella pneumoniae, a Gram-negative pathogen that colonizes mucosal surfaces of the human gastrointestinal and respiratory tracts. Cultured bacteria are exposed to antibiotics, the total proteomes of collected cell pellets are converted to complex peptide mixtures by filter-aided sample preparation (FASP), and the peptides are further processed by an optimized desalting procedure. A mixture of peptides from Klebsiella pneumoniae proteomes are analyzed by high-resolution mass spectrometry (MS) that is coupled to sensitive and comprehensive nano-liquid chromatography (nano-LC). The generic method described here for the identification and quantification of the proteome will provide a snapshot of differential protein abundances resulting from antimicrobial sensitivities, which can be used to model directed perturbations of the global system and to select targets of specific interest for further study.
Keywords: Antibiotic susceptibility; Enterobacteriaceae; Klebsiella pneumoniae; Label-free quantitative proteomics; Mass spectrometry; Proteomic remodeling; Transient phenotypic resistance.