Quantitative Proteomic Analysis of Drug-Induced Changes in Mycobacteria Minerva A. Hughes, † Jeffrey C. Silva, ‡ Scott J. Geromanos, ‡ and Craig A. Townsend* ,† Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore Maryland 21218, and Waters Corporation, 34 Maple Street, Milford Massachusetts 01757-3696 Received July 29, 2005 A new approach for qualitative and quantitative proteomic analysis using capillary liquid chromatog- raphy and mass spectrometry to study the protein expression response in mycobacteria following isoniazid treatment is discussed. In keeping with known effects on the fatty acid synthase II pathway, proteins encoded by the kas operon (AcpM, KasA, KasB, Accd6) were significantly overexpressed, as were those involved in iron metabolism and cell division suggesting a complex interplay of metabolic events leading to cell death. Keywords: isoniazid • tuberculosis • protein profiling • liquid chromatography • mass spectrometry Introduction Tuberculosis (TB) continues to be a major cause of disease and mortality with an estimated 2 million deaths annually. 1 Approximately one-third of the world’s population is thought to be infected with Mycobacterium tuberculosis, the etiological agent of TB. Despite the reported success of directly observed treatments, short course (DOTS), noncompliance is one of the factors leading to the emergence of multi-drug resistant strains (MDR-TB). MDR-TB refers to those strains resistant to two or more of the five first line anti-TB drugs (isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin). Patients infected with an MDR-TB strain have a mortality rate of 60-90%, equivalent to those without treatment. 2 The matter is further complicated by a rapid progression of the disease and increased risk of reactivation of latent TB in individuals co-infected with Human Immunodeficiency Virus (HIV). 3 The growing global burden of TB creates an urgent need to define new classes of therapeutics effective against MDR strains and with improved sterilizing activity. An incomplete understanding of the physiology of myco- bacteria and the mechanisms associated with drug sensitivity has been the greatest impediment toward significant advances in drug-development. Recently the completed genome se- quences for M. tuberculosis H37Rv and other mycobacterial strains have become available. 4-6 They have made it possible to apply existing bioinformatic, genomic, and proteomic techniques to obtain a better understanding of the pathophysi- ology of mycobacteria as well as pathways of heightened sensitivity exploitable for rational drug-discovery efforts. For example, comparative genomic studies using DNA microarray technology have identified ‘core’ genes of the M. tuberculosis complex that could provide highly selective drug targets. 7 Additional gene expression profiling studies offered further insights into the metabolism of M. tuberculosis defining adap- tive responses to intracellular phagocytosis, 8 heat shock, 9 oxidative stress, 10 nutrient depletion, 11 and responses to various chemotherapeutics. 12 Though informative, genomic analysis alone provides only a limited view of the dynamics associated with cellular re- sponses to a particular stimulus or at steady state. A systemic analysis of the proteomic and metabolic fluctuations is needed to complement existing genomic studies. Compared to mRNA studies, proteomic analysis provides a more accurate assess- ment of the conditional changes because the measurement focuses on the functionally relevant species. Furthermore, a direct correlation between mRNA expression and changes in the protein population at either steady-state or in response to a stimulus does not exist due in part to post-translational control mechanisms. 13 Since proteins are the target for most drugs, our understanding of drug-related responses at the level of the proteome will undoubtedly unravel the important dynamics of a drug’s mechanism of action and define new pathways for drug discovery. While our primary interests are in understanding drug related effects in mycobacteria, the methodology applied in this study is applicable to studying a wide array of adaptive responses. Traditionally, quantitative proteomics relied on the resolving power of two-dimensional gel electrophoresis coupled with mass spectrometry (2DE-MS) for qualitative and quantitative protein identification. Despite its popularity, the accuracy of protein quantitation using this method can be ambiguous due to post-translational modifications resulting in multiple spots for a single protein or multiple proteins in a single spot, protein degradation, presence of protein isoforms and variability in protein recovery from in-gel digests. To compensate for the limitations of 2DE-MS, standardized gel-free methods have been developed. They involve the combination of stable- isotope labeling during sample preparation coupled with * To whom correspondence should be addressed. Tel: (410) 516-7444. Fax: (410) 261-1233. E-mail: ctownsend@jhu.edu. † Department of Chemistry, The Johns Hopkins University. ‡ Waters Corporation, Milford, MA. 54 Journal of Proteome Research 2006, 5, 54-63 10.1021/pr050248t CCC: $33.50  2006 American Chemical Society Published on Web 12/02/2005