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Journal of Bacteriology, August 2008, p. 5265-5278, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.00383-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Relative Quantitative Comparisons of the Extracellular Protein Profiles of Staphylococcus aureus UAMS-1 and Its sarA, agr, and sarA agr Regulatory Mutants Using One-Dimensional Polyacrylamide Gel Electrophoresis and Nanocapillary Liquid Chromatography Coupled with Tandem Mass Spectrometry ^,

Richard C. Jones,^2, Joanna Deck,¹ Ricky D. Edmondson,^#,2 and Mark E. Hart^1*

Divisions of Microbiology,¹ Systems Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079-9502²

Received 17 March 2008/ Accepted 25 May 2008

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances.

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* Corresponding author. Mailing address: Division of Microbiology (HFT-250), National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079-9502. Phone: (870) 543-7625. Fax: (870) 543-7307. E-mail: mark.hart{at}fda.hhs.gov

Published ahead of print on 6 June 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: NextGen/PRS, Ann Arbor, MI 48108.

^# Present address: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR 72305.

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Journal of Bacteriology, August 2008, p. 5265-5278, Vol. 190, No. 15
0021-9193/08/$08.00+0     doi:10.1128/JB.00383-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.