Coxiella burnetiiWhole Cell Lysate Protein Identification by Mass Spectrometry and Tandem Mass Spectrometry

Abstract

The whole cell lysate of Coxiella burnetii strain RSA 493 was separated by two-dimensional electrophoresis and more than 500 protein spots were found on silver-stained reference map. Spots from the gels were subjected to identification based on peptide mass fingerprinting (PMF). In order to identify additional proteins, tandem mass spectrometry (MS/MS) using electrospray and matrix-assisted laser desorption/ionization techniques was applied. The three independent approaches resulted in the identification of 197 open reading frames (ORFs). Fifty-two proteins were identified by PMF and at least with one of the MS/MS methods, 37 proteins with both MS/MS instruments, and 19 proteins with all three techniques applied. All predicted C. burnetii ORFs were compared with the Clusters of Orthologous Groups database. The data related to identified proteins were stored and indexed in a file that can be read and searched using Microsoft Access.

Proteomic comparison of virulent phase I and avirulent phase II of Coxiella burnetii, the causative agent of Q fever

“…Additionally, 150 proteins not reported in previous proteomic studies of C. burnetii [40][41][42][43][44][45][46] were identified in this study (Supplemental Table 3). Among these are a number of proteins potentially associated with pathogenicity and virulence.…”

“…Proteomic studies that incorporate an electrophoresisbased strategy typically use either 1DE or 2DE. Previous proteomic studies of C. burnetii have included 2DE separations [40][41][42][43]46]. Using only this strategy, however, makes it difficult to detect low-abundance and/or very basic proteins.…”

“…The methyltransferases are candidate enzymes for synthesis Vir and Strep in C. burnetii in phase I [39,41]. It is likely that the S-adenosyl-dependent methyl transferase (Q83DL9) is part of the Vir biosynthetic pathway [35].…”

“…To date nearly 200 C. burnetii proteins have been documented in the literature, including some from both phase I and II cells [40][41][42][43][44][45][46]. Unfortunately analysis of the two phases has typically been done under different conditions, complicating comparisons.…”

“…Growth of C. burnetii was monitored by Gimenez staining [48], and electron microscopy was used to assess the quality of the purified fractions. [41], and proteins were precipitated from a mixture of chloroform, methanol, and water [49]. Insoluble matter was removed by centrifugation for 20 min at 14,000 g and 4°C.…”