Purification and identification of trichloroethylene induced proteins from Stenotrophomonas maltophilia PM102 by immuno-affinity-chromatography and MALDI-TOF Mass spectrometry

Piyali Mukherjee,Pranab Roy

doi:10.1186/2193-1801-2-207

Abstract

A novel bacterial isolate capable of growth on trichloroethylene as the sole carbon source was identified as Stenotrophomonas maltophilia PM102 by 16S rDNA sequencing (GenBank Acc.no. JQ797560). Serum was obtained from a rabbit immunized with the total protein extracted from the PM102 isolate grown in 0.2% TCE with 0.2% peptone. Antibodies to the common antigens were removed by preadsorbing the serum antibody on total protein extracted from the PM102 strain grown in 0.2% peptone. Western blot with the preadsorbed antibody reacted to a single band in TCE and TCE with peptone lane. No reaction was seen in peptone lane. This preadsorbed antibody specific for TCE inducible antigens was immobilised on epoxy activated sepharose 6B and total protein from PM102 cells grown in minimal medium with TCE as the sole carbon source was purified through the column. The bound protein fraction was eluted and resolved through 12% SDS PAGE. Four prominent bands observed in the protein profile were analysed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MS) and tandem mass spectrometry (MS/MS) after in gel digestion with 25 ng/μl trypsin. A number of mono/di-oxygenases that cometabolise TCE in presence of some other primary carbon source are present in literature but this is the first attempt in identification of TCE induced proteins linked to metabolic activity with oxidoreductase like function, from a bacterial isolate that utilises TCE as the sole carbon source.

Highlights

The health effects of Trichloroethylene are a complete adversary to its sweet odour and taste (Gist & Burg 1995)
P1 was matched to a putative phosphormannomutase from Streptomyces avermitilis MA-4680 with accession number: (Figure 7a); P2 closest match was a putative monooxygenase from Streptomyces coelicolor A3 (Figure 7b); P3 was matched to a hypothetical protein from Bacillus licheniformis.(Q65KM5_BACLI) (Figure 7c) and P4 closest match was a putative 6phosphogluconolactonase from Streptomyces coelicolor A3 (Figure 7d)
Many protein bands were obtained with the crude protein extracted from PM102 cells grown in minimal medium with 0.2% TCE as the sole carbon source but after immune purification of this crude protein, only four bands were detected in the SDS profile

Summary

Introduction

The health effects of Trichloroethylene are a complete adversary to its sweet odour and taste (Gist & Burg 1995). (PM 102 isolate) that grows on TCE as the sole carbon source (Mukherjee & Roy 2012). Studies involving identification of the proteins employed by the PM102 strain in the degradation of TCE necessitated the extraction of these proteins in a highly purified form. The principle of protein identification using peptide mass fingerprinting (PMF) is based on the comparison of a set of experimental peptide masses obtained by trypsin digestion, with a database containing in silico digested peptide masses of known proteins (Thiede et al 2005; Pappin et al 1993). Till date no information is available at the molecular level on proteomic compositions of bacteria capable of growth on TCE as the sole carbon source. We employed immunoproteomics technology to purify and identify the TCE induced proteins from the PM102 isolate that utilizes TCE as the primary carbon source

Methods

Results

Conclusion