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Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis

Abstract

The aim of this study was to examine the protein expression profiles of persistentChlamydia pneumoniae by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Although 2D PAGE is still the method of choice for separating and detecting components of complex protein mixtures, it has several distinct disadvantages; i.e., being labor-intensive and having a bias toward proteins within the dynamic range of the gel condition. Hence, SELDI-TOF-MS technology was used to complement 2D PAGE.C. pneumoniae-infected HEp2 cells were treated with or without IFN-γ, and protein expression profiles were determined at 48 h postinfection (hpi). Unfractionated monolayers were also used for protein profiling by SELDI-TOF, using two different chip surface types: weak cation exchanger and hydrophobic surface. Under IFN-γ-induced persistence,C. pneumoniae expresses an altered protein expression profile. Twenty chlamydial proteins showed differential regulatory patterns by SELDI-TOF-MS, two of which, HSP-70 cofactor, and a hypothetical protein, were identified by 2D PAGE and mass spectrometry. Two additional proteins, phosphatidylserine decarboxylase and 30S ribosomal protein S17, were exclusively identified by SELDI TOF-MS analysis, as these were not present in sufficient quantity for detection by 2D PAGE. We propose that a combination of 2D-PAGE and SELDI-TOF-MS may complement the disadvantages of each technique alone and may provide a rapid and precise screening technique.

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Correspondence to Sanghamitra Mukhopadhyay or James T. Summersgill.

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Key Words

  • Proteomics
  • SELDI-TOF-MS
  • 2D gel electrophoresis
  • C. pneumoniae