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Applied and Environmental Microbiology, September 2007, p. 5653-5656, Vol. 73, No. 17
0099-2240/07/$08.00+0 doi:10.1128/AEM.00667-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Labeling of Bifidobacterium longum Cells with 13C-Substituted Leucine for Quantitative Proteomic Analyses
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Yohann Couté,1
Céline Hernandez,2
Ron D. Appel,2
Jean-Charles Sanchez,1 and
Abelardo Margolles1,3*
Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, University of Geneva, 1 Rue Michel Servet, 1211 Geneva 14, Switzerland,1
Proteome Informatics Group, Swiss Institute of Bioinformatics, Geneva, Switzerland,2
Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas (CSIC), Ctra. Infiesto s/n, 33300 Villaviciosa, Asturias, Spain3
Received 23 March 2007/
Accepted 25 June 2007
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ABSTRACT
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Stable isotope labeling of amino acids in cell culture was used for Bifidobacterium longum. A comprehensive proteomic strategy was developed and validated by designing an appropriate semidefined medium that allows stable replacement of natural leucine by [13C6]leucine. Using this strategy, proteins having variations of at least 50% in their expression rates can be quantified with great confidence.
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INTRODUCTION
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Bifidobacteria are anaerobic bifid or multiply branching gram-positive rods that constitute one of the most numerous populations in the gastrointestinal tract of humans. Particularly in breast-feed infants, they can represent up to 91% of the total gut microbiota, and Bifidobacterium longum is one of the most representative species (3). Today, some strains of the genus Bifidobacterium are considered to be probiotics on the basis of their role in promoting beneficial health effects (12). However, the mechanistic events underlying their mode of action remain to be solidly established (6).
The genome sequencing of B. longum NCC2705 (15) has recently prompted some investigations at the proteomic level. Two-dimensional electrophoresis and multidimensional chromatography separation techniques coupled to protein identification by mass spectrometry have provided insights into the changes in protein abundance underlying basic biological processes (13, 14, 19). However, these approaches are not always appropriate for studying specific components of the proteome and subcellular fractions, such as low-abundance proteins, proteins with extreme pI values and Mr, and very hydrophobic proteins, including membrane proteins. Although this problem could be partially solved by using multidimensional chromatography, this method does not allow quantification of differential protein production under different physiological conditions (18). With the aim of overcoming the problems mentioned above, we developed a method based on the selective incorporation of isotopically labeled leucine into Bifidobacterium cells. B. longum NCIMB8809, a human isolate with potential probiotic activity, was chosen as a model microorganism for this study (10).
Stable isotope labeling of amino acids in cell cultures (SILAC) is a simple and accurate procedure that can be used as a quantitative proteomic approach with many growing eukaryotic cell types (5). It is based on a comparison of the protein levels in cells grown in two formulations of the same medium that differ only by the fact that one formulation contains a nonradioactive, isotopically labeled form of an amino acid (9). By measuring the ratio of light peptides to heavy peptides, the relative abundance of proteins from cultures treated under different conditions can be determined. Recently, this method was successfully adapted for Escherichia coli. Neher and coworkers constructed a mutant with a disrupted leuB gene, the gene coding for the enzyme responsible for the last step in leucine biosynthesis. Using the SILAC strategy, they obtained complete incorporation of [13C]leucine in their cells (7). However, the lack of efficient molecular techniques for disrupting genes strongly limits functional studies of Bifidobacterium (17). In the present work we tackled this challenge by defining a medium that allows growth of B. longum NCIMB8809 with high-level, stable incorporation of [13C6]leucine. Incorporation of [13C6]leucine (containing six 13C atoms) into a protein or peptide leads to a 6-Da shift in the molecular mass due to the labeled leucine compared to the protein or peptide that contains natural leucine.
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Formulation of a semidefined medium.
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Two different chemically defined media for Lactobacillus species have been described previously (4, 11). Based on the formulations of these media, we designed a semidefined medium which includes the permeate resulting from dialysis (with a 2-kDa-cutoff membrane) of MRS medium (2) (semidefined medium for B. longum [SDMBL] [see the supplemental material]). The ability of B. longum NCIMB8809 to grow in this broth was tested. Cells were able to reach optical densities at 600 nm of up to 1.4. For our analysis, all the cultures were harvested in the exponential phase of growth at optical densities at 600 nm between 0.3 and 0.8. In this range, a maximum specific growth rate (µ) of 0.54 h–1 was achieved. The growth rate was estimated from the growth curve by fitting the data to the equation Nt =N0 x eµ x t, where Nt and N0 are the cell densities at time t and time zero in the exponential growth phase, respectively. This medium was used for further development.
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Stable incorporation of [13C6]leucine in B. longum NCIMB8809 cells.
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Leucine was chosen for these experiments because, together with valine, it is one of the most highly represented amino acids in the B. longum proteins (www.cbs.dtu.dk/services/GenomeAtlas). The efficiency of incorporation into B. longum of [13C6]leucine present in SDMBL was tested. For this, cultures in MRSC (MRS supplemented with 0.05% L-cysteine) were grown overnight under standard conditions (13), washed three times in SDMBL, and resuspended in this medium. Cells were grown for 5.8, 9.3, 13.1, or 16.0 generations (the time interval needed for doubling of the optical density was considered 1 generation). In each case, the inoculum used was diluted to allow harvesting of the cells in the exponential phase of growth. Cultures grown in SDMBL with normal leucine were used as a negative incorporation control. We found that the growth of cells in SDMBL was the same independent of the form of leucine used, as shown by the growth rates, indicating that the presence of the heavy amino acid does not influence the growth parameters (data not shown).
To determine the time course and rate of incorporation of [13C6]leucine, cell-free protein extracts at the indicated generations from the cultures mentioned above were prepared by sonication, as previously described (8). Proteins were run on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels, and three different fractions (Fig. 1) were digested in the gels as described by Scherl et al. (16). The peptides obtained were analyzed with a 4700 matrix-assisted laser desorption ionization—time of flight/time of flight mass analyzer (1). As shown in Fig. 2, incorporation of [13C6]leucine was readily detectable in peptides after 5.8 generations. The maximum incorporation was observed at 9.3 generations, and the incorporation remained very stable up to 16 generations (Fig. 3). For peptides containing one leucine, this was shown by the presence of peaks at a mass that was 6 Da higher than the mass of the natural peptide. The maximum incorporation rate was 85% ± 2%. For peptides containing two leucines (glutamine synthetase 1 and glucose-6-phosphate isomerase in Fig. 2), two peaks were observed for 9.3 generations: one containing two [13C6]leucines (with a mass 12 Da higher than the mass of the natural peptide) and the other (intermediate peak) containing one natural leucine and one [13C6]leucine (with a mass 6 Da higher than the mass of the natural peptide). Peptides containing only natural leucine were nearly undetectable. The incorporation profiles were very similar independent of the peptide analyzed. Therefore, [13C6]leucine incorporation is stable in B. longum cells after 9.3 generations, indicating that cells can be adapted for use in SILAC experiments to quantitate protein levels.
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FIG. 1. SDS-PAGE profile of cell-free protein extracts from B. longum NCIMB8809. The arrows indicate the positions of the fractions selected for mass spectrometry analyses. Lane kDa contained molecular mass standards.
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FIG. 2. Incorporation of [13C6]leucine at various time points. Representative mass spectra for peptides resulting from digestion of proteins from fraction 1 are shown. The arrows indicate the presence of the heavy and intermediate peptide forms, and the bold numbers indicate the percentages of the remaining light peptide form. The proteins indicated in parentheses were identified according to the clusters of orthologous genes for B. longum NCC2705. G, generations.
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FIG. 3. Stable incorporation of [13C6]leucine in B. longum cells. The bars indicate the percentages of light amino acid after growth of the cells for 0 (a), 5.8 (b), 9.3 (c), 13.1 (d), and 16.0 (e) generations for peptides containing one leucine (black bars) or two leucines (gray bars). Three independent experiments were carried out, and the results are the averages for 17 peptides containing one leucine and 12 peptides containing two leucines identified with great confidence (P < 1.0E–6) from fraction 1. The error bars indicate the standard deviations.
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Validation of the method for quantitative proteomics.
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Two mixing experiments were performed using known protein concentrations of both light and heavy cell cultures grown for 13.12 ± 0.26 generations. Protein extracts were quantified using the Bradford method as previously described (13), mixed at theoretical ratios of 1:1, 2:1, 1:2, 5:1, and 1:5, separated by SDS-PAGE (Fig. 1), digested in the gel, and analyzed by tandem mass spectrometry. Proteins were accurately identified using Phenyx software (Genebio, Geneva, Switzerland). Experimental ratios for identified peptides were calculated from isotope cluster areas using software developed in-house (www.ProteomeCommons.org). This software was created using the Visual Basic language (Microsoft) in order to make it easy to manage and interact with Excel files (Microsoft). It uses as input a specific Excel output file produced by the Phenyx identification software. The parameters for the labeled amino acid used, the mass shift created by the modified amino acid, and the error tolerance for the peptide mass between light and heavy forms have to be chosen by the user. In our case, the software was run with an error of 0.2 Da and a mass shift of 6 Da per leucine. Besides its basic functionalities the software was adapted for our specific purposes. Since, as described above, the incorporation of [13C6]leucine was not total in heavy cell cultures, the software was modified to correct for the effect of the presence of the light amino acid in the samples. For peptides containing one leucine, a correction of 15% was applied to the areas of the light forms. For peptides containing several leucines, the areas of the intermediate peaks were added to the area of the heavy form. Using this approach, we were able to accurately quantitate B. longum proteins using SILAC (Table 1; see Table S1 in the supplemental material). The average coefficient of variation of these experiments was about 20% (maximum, 30.2%), indicating that applying SILAC to B. longum allowed us to reliably quantify proteins with 50% variation in their expression rates.
In conclusion, the method developed in this work can be satisfactorily used for quantitative proteomic studies of B. longum. It can be adapted to any bacteria which allow stable incorporation of isotopically labeled amino acids in a defined medium. Furthermore, this method does not require peptide labeling steps, and several peptides from the same protein can be compared, minimizing the amount of material needed and reinforcing quantitation data.
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ACKNOWLEDGMENTS
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This work was financed by European Union FEDER funds and the Spanish Plan Nacional de I + D (project AGL2004-06727-C02-01/ALI and fellowship PR2006-0421) and by the Swiss National Science Foundation (grant 3100-A0-104214).
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FOOTNOTES
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* Corresponding author. Mailing address: Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas (CSIC), Ctra. Infiesto s/n, 33300 Villaviciosa, Asturias, Spain. Phone: 34 985 89 21 31. Fax: 34 985 89 22 33. E-mail: amargolles{at}ipla.csic.es 
Published ahead of print on 29 June 2007.
Supplemental material for this article may be found at http://aem.asm.org/.
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Applied and Environmental Microbiology, September 2007, p. 5653-5656, Vol. 73, No. 17
0099-2240/07/$08.00+0 doi:10.1128/AEM.00667-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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ABSTRACT
INTRODUCTION
