Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 13

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Proteomic Analysis of Proteins Secreted
by Streptococcus pyogenes
Michelle A. Chaussee, Emily J. McDowell, and Michael S. Chaussee
Summary
Streptococcus pyogenes secretes various proteins to the extracellular environment.
During infection, these proteins interact with human macromolecules and contribute to
pathogenesis. We describe a proteomic approach routinely used in our laboratory to charac-
terize culture supernatant proteins using small-format two-dimensional gel electrophoresis.
Proteins are collected after overnight growth of the bacteria in broth media. Compounds
that inhibit isoelectric focusing, such as salts, are removed by enzymatic treatment and
precipitation with trichloroacetic acid and acetone. Following resuspension in denaturing
solution, the proteins are separated by isoelectric focusing using a 7-cm immobilized
strip with a pH gradient of 4-7. Subsequently, proteins are further separated with
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with
SYPRO Ruby. The small-gel format requires less time, reagents, and smaller culture
volumes compared with large-format approaches, while still resolving and detecting a
large proportion of the exoprotein fraction.
Key Words: Proteomics; exoprotein; Streptococcus pyogenes ; two-dimensional gel
electrophoresis.
1. Introduction
Streptococcus pyogenes secretes many proteins that interact with human
macromolecules (1,2) . Not surprisingly, many are important determinants of
host-pathogen interactions and contribute to virulence. In vitro, the secreted
proteins can be readily isolated from culture supernatants. Extracellular proteins
include both those with type II signal secretion signals and (for reasons