Labeling of Bifidobacterium longum Cells with 13C-Substituted Leucine for Quantitative Proteomic Analyses

Stable isotope labeling of amino acids in cell culture was usedfor Bifidobacterium longum. A comprehensive proteomic strategywas developed and validated by designing an appropriate semidefinedmedium that allows stable replacement of natural leucine by[¹³C₆]leucine. Using this strategy, proteins having variationsof at least 50% in their expression rates can be quantifiedwith great confidence.

INTRODUCTION

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INTRODUCTION

Bifidobacteria are anaerobic bifid or multiply branching gram-positiverods that constitute one of the most numerous populations inthe gastrointestinal tract of humans. Particularly in breast-feedinfants, they can represent up to 91% of the total gut microbiota,and Bifidobacterium longum is one of the most representativespecies (3). Today, some strains of the genus Bifidobacteriumare considered to be probiotics on the basis of their role inpromoting beneficial health effects (12). However, the mechanisticevents underlying their mode of action remain to be solidlyestablished (6).

The genome sequencing of B. longum NCC2705 (15) has recentlyprompted some investigations at the proteomic level. Two-dimensionalelectrophoresis and multidimensional chromatography separationtechniques coupled to protein identification by mass spectrometryhave provided insights into the changes in protein abundanceunderlying basic biological processes (13, 14, 19). However,these approaches are not always appropriate for studying specificcomponents of the proteome and subcellular fractions, such aslow-abundance proteins, proteins with extreme pI values andM_r, and very hydrophobic proteins, including membrane proteins.Although this problem could be partially solved by using multidimensionalchromatography, this method does not allow quantification ofdifferential protein production under different physiologicalconditions (18). With the aim of overcoming the problems mentionedabove, we developed a method based on the selective incorporationof isotopically labeled leucine into Bifidobacterium cells.B. longum NCIMB8809, a human isolate with potential probioticactivity, 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 quantitativeproteomic approach with many growing eukaryotic cell types (5).It is based on a comparison of the protein levels in cells grownin two formulations of the same medium that differ only by thefact that one formulation contains a nonradioactive, isotopicallylabeled form of an amino acid (9). By measuring the ratio oflight peptides to heavy peptides, the relative abundance ofproteins from cultures treated under different conditions canbe determined. Recently, this method was successfully adaptedfor Escherichia coli. Neher and coworkers constructed a mutantwith a disrupted leuB gene, the gene coding for the enzyme responsiblefor the last step in leucine biosynthesis. Using the SILAC strategy,they obtained complete incorporation of [¹³C]leucine in theircells (7). However, the lack of efficient molecular techniquesfor disrupting genes strongly limits functional studies of Bifidobacterium(17). In the present work we tackled this challenge by defininga 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 toa 6-Da shift in the molecular mass due to the labeled leucinecompared to the protein or peptide that contains natural leucine.

Formulation of a semidefined medium.

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Formulation of a semidefined...

Two different chemically defined media for Lactobacillus specieshave been described previously (4, 11). Based on the formulationsof these media, we designed a semidefined medium which includesthe 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 NCIMB8809to grow in this broth was tested. Cells were able to reach opticaldensities at 600 nm of up to 1.4. For our analysis, all thecultures were harvested in the exponential phase of growth atoptical densities at 600 nm between 0.3 and 0.8. In this range,a maximum specific growth rate (µ) of 0.54 h^–1 wasachieved. The growth rate was estimated from the growth curveby fitting the data to the equation N_t =N₀ x e^{µ x t}, whereN_t and N₀ are the cell densities at time t and time zero inthe exponential growth phase, respectively. This medium wasused for further development.

Stable incorporation of [¹³C₆]leucine in B. longum NCIMB8809 cells.

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Stable incorporation of...

Leucine was chosen for these experiments because, together withvaline, it is one of the most highly represented amino acidsin the B. longum proteins (www.cbs.dtu.dk/services/GenomeAtlas).The efficiency of incorporation into B. longum of [¹³C₆]leucinepresent in SDMBL was tested. For this, cultures in MRSC (MRSsupplemented with 0.05% L-cysteine) were grown overnight understandard conditions (13), washed three times in SDMBL, and resuspendedin this medium. Cells were grown for 5.8, 9.3, 13.1, or 16.0generations (the time interval needed for doubling of the opticaldensity was considered 1 generation). In each case, the inoculumused was diluted to allow harvesting of the cells in the exponentialphase of growth. Cultures grown in SDMBL with normal leucinewere used as a negative incorporation control. We found thatthe growth of cells in SDMBL was the same independent of theform of leucine used, as shown by the growth rates, indicatingthat the presence of the heavy amino acid does not influencethe 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 fromthe cultures mentioned above were prepared by sonication, aspreviously described (8). Proteins were run on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels,and three different fractions (Fig. 1) were digested in thegels as described by Scherl et al. (16). The peptides obtainedwere analyzed with a 4700 matrix-assisted laser desorption ionization—timeof flight/time of flight mass analyzer (1). As shown in Fig.2, incorporation of [¹³C₆]leucine was readily detectable inpeptides after 5.8 generations. The maximum incorporation wasobserved at 9.3 generations, and the incorporation remainedvery stable up to 16 generations (Fig. 3). For peptides containingone leucine, this was shown by the presence of peaks at a massthat was 6 Da higher than the mass of the natural peptide. Themaximum incorporation rate was 85% ± 2%. For peptidescontaining two leucines (glutamine synthetase 1 and glucose-6-phosphateisomerase in Fig. 2), two peaks were observed for 9.3 generations:one containing two [¹³C₆]leucines (with a mass 12 Da higherthan the mass of the natural peptide) and the other (intermediatepeak) containing one natural leucine and one [¹³C₆]leucine (witha mass 6 Da higher than the mass of the natural peptide). Peptidescontaining only natural leucine were nearly undetectable. Theincorporation profiles were very similar independent of thepeptide analyzed. Therefore, [¹³C₆]leucine incorporation isstable in B. longum cells after 9.3 generations, indicatingthat cells can be adapted for use in SILAC experiments to quantitateprotein 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.

Validation of the method for quantitative proteomics.

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Validation of the method...

Two mixing experiments were performed using known protein concentrationsof both light and heavy cell cultures grown for 13.12 ±0.26 generations. Protein extracts were quantified using theBradford method as previously described (13), mixed at theoreticalratios 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 peptideswere calculated from isotope cluster areas using software developedin-house (www.ProteomeCommons.org). This software was createdusing the Visual Basic language (Microsoft) in order to makeit easy to manage and interact with Excel files (Microsoft).It uses as input a specific Excel output file produced by thePhenyx identification software. The parameters for the labeledamino acid used, the mass shift created by the modified aminoacid, and the error tolerance for the peptide mass between lightand 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 shiftof 6 Da per leucine. Besides its basic functionalities the softwarewas adapted for our specific purposes. Since, as described above,the incorporation of [¹³C₆]leucine was not total in heavy cellcultures, the software was modified to correct for the effectof the presence of the light amino acid in the samples. Forpeptides containing one leucine, a correction of 15% was appliedto the areas of the light forms. For peptides containing severalleucines, the areas of the intermediate peaks were added tothe area of the heavy form. Using this approach, we were ableto accurately quantitate B. longum proteins using SILAC (Table1; see Table S1 in the supplemental material). The average coefficientof variation of these experiments was about 20% (maximum, 30.2%),indicating that applying SILAC to B. longum allowed us to reliablyquantify proteins with 50% variation in their expression rates.

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TABLE 1. Quantitation of peptides^a

In conclusion, the method developed in this work can be satisfactorilyused for quantitative proteomic studies of B. longum. It canbe adapted to any bacteria which allow stable incorporationof isotopically labeled amino acids in a defined medium. Furthermore,this method does not require peptide labeling steps, and severalpeptides from the same protein can be compared, minimizing theamount of material needed and reinforcing quantitation data.

ACKNOWLEDGMENTS

This work was financed by European Union FEDER funds and theSpanish Plan Nacional de I + D (project AGL2004-06727-C02-01/ALIand fellowship PR2006-0421) and by the Swiss National ScienceFoundation (grant 3100-A0-104214).

FOOTNOTES

* 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/.

REFERENCES

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