Sulfur-34S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34) of Proteomic Changes in Pseudomonas fluorescens during Naphthalene Degradation

Florian-Alexander Herbst,Martin Taubert,Nico Jehmlich,Tobias Behr,Frank Schmidt,Martin Von Bergen,Jana Seifert

doi:10.1074/mcp.m112.025627

Abstract

The relative quantification of proteins is one of the major techniques used to elucidate physiological reactions. Because it allows one to avoid artifacts due to chemical labeling, the metabolic introduction of heavy isotopes into proteins and peptides is the preferred method for relative quantification. For eukaryotic cells, stable isotope labeling by amino acids in cell culture (SILAC) has become the gold standard and can be readily applied in a vast number of scenarios. In the microbial realm, with its highly versatile metabolic capabilities, SILAC is often not feasible, and the use of other (13)C or (15)N labeled substrates might not be practical. Here, the incorporation of heavy sulfur isotopes is shown to be a useful alternative. We introduce (34)S stable isotope labeling of amino acids for quantification and the corresponding tools required for spectra extraction and disintegration of the isotopic overlaps caused by the small mass shift. As proof of principle, we investigated the proteomic changes related to naphthalene degradation in P. fluorescens ATCC 17483 and uncovered a specific oxidative-stress-like response.

Highlights

From the ‡UFZ - Helmholtz Centre for Environmental Research, Department of Proteomics, Permoserstrasse 15, 04318 Leipzig, Germany; §Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, D-79104 Freiburg, Germany; ¶School of Environmental Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom; ʈDepartment of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Strasse 15a, 17487 Greifswald, Germany; **Hochschule Wismar - University of Applied Sciences: Technology, Business and Design, Philipp-Muller-Strasse 14, 23966 Wismar, Germany; ‡‡Department of Metabolomics, UFZ - Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany; §§Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, 9000 Aalborg, Denmark; ¶¶Institute of Animal Nutrition, University of Hohenheim, 70599 Stuttgart, Germany
Because the mass shift caused by the replacement of two 12C atoms with two 13C atoms and that caused the replacement of one 32S atom with its 34S isotope differ by only 0.011 Da, a high mass resolution is required in order to distinguish between these two types of incorporation [9]
(13)— have been studied in detail in Pseudomonades, no transcriptomic or global proteomic study has been performed. In this proteomics study utilizing 34S stable isotope labeling of amino acids for quantification (SULAQ34), naphthalene was used as a model low-molecular-weight polycyclic aromatic hydrocarbon (PAH) to investigate the PAH metabolism in P. fluorescens and enhance our understanding with regard to physiological adaptation

Summary

EXPERIMENTAL PROCEDURES

Cultivation—Preparatory cultures of P. fluorescens ATCC 17483 were grown in 5 ml mineral medium (760 mg/l NH4Cl, 680 mg/l KH2PO4, 871 mg/l K2HPO4, 5.5 mg/l CaCl2x6H2O, 0.25 mg/l Na2MoO4x4H2O, pH 7.0) supplemented with trace element solution SL-10 (1 ml/l)(14, 15), MgCl2x6H2O (1 mM), and sodium succinate (10 mM) as the carbon source. After 6 min, the peptides were eluted into a separation column (nanoAcquity UPLC column, C18, 75 ␮m ϫ 15 cm, 1.75 ␮m, Waters). Using a nano-high pressure liquid chromatography system (nanoAcquity UPLC, Waters) coupled to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific), the SULAQ34 peptides were eluted. PeakExtractor (see supplemental PeakExtractor.zip) was used to extract all MS1 spectra belonging to one peptide as defined in a list containing peptide identification information comprising the peptide sequence, peptide ID, mass, retention time or scan number, and charge. The final log ratio per protein was the mean of the different replicate values. Maxquant LFQ values were averaged for naphthalene and sodium succinate samples and processed analogously to the labeled approach

RESULTS AND DISCUSSION

SV ϭ 1 Cold shock protein GN ϭ capB PE ϭ 3

CONCLUSIONS