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Antimicrobial Agents and Chemotherapy, November 2001, p. 3046-3055, Vol. 45, No. 11
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.11.3046-3055.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Single Ribosomal Protein Mutations in Antibiotic-Resistant Bacteria Analyzed by Mass Spectrometry

Sheri K. Wilcox,1,dagger Gregory S. Cavey,2 and James D. Pearson1,*

Departments of Protein Science1 and Structural, Analytical, and Medicinal Chemistry,2 Pharmacia Corporation, Kalamazoo, Michigan 49007

Received 16 April 2001/Returned for modification 1 June 2001/Accepted 16 August 2001

Mutations in several ribosomal proteins are known to be related to antibiotic resistance. For several strains of Escherichia coli, the mutated protein is known but the amino acid actually altered has not been documented. Characterization of these determinants for antibiotic resistance in proteins will further the understanding of the precise mechanism of the antibiotic action as well as provide markers for resistance. Mass spectrometry can be used as a valuable tool to rapidly locate and characterize mutant proteins by using a small amount of material. We have used electrospray and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to map out all 56 ribosomal proteins in E. coli based on intact molecular masses. We used this fingerprinting approach to locate variants of ribosomal proteins displaying a change in mass. In particular we have studied proteins responsible for streptomycin, erythromycin, and spectinomycin resistance in three strains of E. coli, and then we characterized each mutation responsible for resistance by analyzing tryptic peptides of these proteins by using MALDI-TOF and nanoelectrospray tandem mass spectrometry. The results provided markers for antibiotic resistance and demonstrated that mass spectrometry can be used to rapidly investigate changes in individual proteins from a complex with picomole amounts of protein.

* Corresponding author. Mailing address: Pharmacia Corporation, Department of Protein Science, 7240-209-317, 301 Henrietta St., Kalamazoo, MI 49007. Phone: (616) 833-8872. Fax: (616) 833-2262. E-mail: james.d.pearson{at}pharmacia.com.

dagger Present address: SomaLogic Inc., Boulder, CO 80301.

Antimicrobial Agents and Chemotherapy, November 2001, p. 3046-3055, Vol. 45, No. 11
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.11.3046-3055.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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