Proteome Analysis of Myxococcus xanthus by Off-Line Two-Dimensional Chromatographic Separation Using Monolithic Poly-(styrene-divinylbenzene) Columns Combined with Ion-Trap Tandem Mass Spectrometry Christian Schley,† Matthias O. Altmeyer,‡ Remco Swart,§ Rolf Mu1 ller,*,‡ and Christian G. Huber*,† Department of Chemistry, Instrumental Analysis and Bioanalysis and Department of Pharmacy, Pharmaceutical Biotechnology, Saarland University, 66123 Saarbru ¨ cken, Germany, and LCPackings-A Dionex Company, Abberdaan 114, 1046 AA Amsterdam, The Netherlands Received May 24, 2006
Myxobacteria are potent producers of secondary metabolites exhibiting diverse biological activities and pharmacological potential. The proteome of Myxococcus xanthus DK1622 was characterized by two-dimensional chromatographic separation of tryptic peptides from a lysate followed by tandem mass spectrometric identification. The high degree of orthogonality of the separation system employing polymer-based strong cation-exchange and monolithic reversed-phase stationary phases was clearly demonstrated. Upon automated database searching, 1312 unique peptides were identified, which were associated with 631 unique proteins. High-molecular polyketide synthetases and nonribosomal peptide synthetases, known to be involved in the biosynthesis of various secondary metabolites, were readily detected. Besides the identification of gene products associated with the production of known secondary metabolites, proteins could also be identified for six gene clusters, for which no biosynthetic product has been known so far. Keywords: shotgun proteome analysis • multidimensional separation • tandem mass spectrometry • Myxococcus xanthus • monolithic columns
1. Introduction Microorganisms producing biologically active secondary metabolites as potential drug candidates play an important role in the pharmaceutical and biotechnological industry.1,2 However, relatively few bacteria are good producers of secondary metabolites, and only two new bacterial groups have been recently added to the producers known already 50 years ago: cyanobacteria and myxobacteria.3 The main goal of research and development in this field is the identification of new and modified natural products with biological activity, as well as optimization of their biosynthesis by biotechnological processes. Myxobacterial secondary metabolites, such as myxovirescins, epothilones, or myxothiazols, are frequently hybrid structures derived from the assembly of carboxylic- and amino acids by polyketide synthetases (PKS) and nonribosomal pep* Corresponding authors. Prof. Dr. Christian Huber, Instrumental Analysis and Bioanalysis, Saarland University, P.O. Box 151150, 66041 Saarbru ¨ cken, Germany. Tel., +49 681 302 2433; fax, +49 681 302 2963; e-mail,
[email protected]. Prof. Dr. Rolf Mu ¨ ller, Pharmaceutical Biotechnology, P.O. Box 151150, 66041 Saarbru¨cken, Germany. Tel., +49 681 302 5474; fax, +49 681 302 5473; e-mail,
[email protected]. † Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University. ‡ Department of Pharmacy, Pharmaceutical Biotechnology, Saarland University. § LCPackings-A Dionex Company.
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tide synthetases (NRPS),4,5 which correspond well with the high number of biosynthetic gene clusters for PKS and/or NRPS found in the genomes of myxobacteria.4,6 Analysis of the proteome of these organisms can not only help to get a better understanding of the biosynthesis of these secondary metabolites and its regulation, but also support the identification of previously unknown compounds based on the expression of gene products that are predicted to be responsible for the production of currently unknown secondary metabolites. Because of the extremely high complexity of proteomes and the wide dynamic range (1-108) of the protein concentrations, their analysis requires methods with high resolving power, high peak capacity, and low limits of detection. The practice of largescale proteome analysis has undergone an enormous development during the past years, mainly due to revolutionary progress in high-throughput protein identification by mass spectrometry (MS) or tandem mass spectrometry (MS/MS) in combination with computer-aided algorithms for rapid and automated data evaluation.7-10 The enormous number of proteins present in proteomes necessitates highly sophisticated separation schemes, mostly including the on-line or off-line combination of two or even more, ideally orthogonal separation stages.11,12 In the top-down approach of proteome analysis,13 two-dimensional separation of the proteome isolated from a biological sample is accomplished at the intact protein level 10.1021/pr0602489 CCC: $33.50
2006 American Chemical Society
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Proteome Analysis of Myxococcus xanthus
followed by their identification by means of protein digestion and fingerprinting methods such as peptide mass fingerprinting (PMF) or peptide fragment fingerprinting (PFF) employing MS or MS/MS.14 Nevertheless, the fractionation of complex mixtures of intact proteins is challenging because of the wide range of physical and chemical properties associated with the individual proteins. Moreover, two-dimensional gel electrophoresis (2D-GE), the most frequently employed separation technology for top-down proteomic analysis, fails to detect very large and/ or hydrophobic proteins15 such as PKS and NRPS, that easily reach molecular masses even higher than 500 kDa.16-19 To tackle these problems, bottom-up proteomic analysis has been devised,13 involving proteolytic digestion of the proteins immediately after their isolation from the cells or tissue to yield a very complex, but relatively homogeneous mixture of proteolytic peptides. One of the milestones in large-scale proteomic analysis was the implementation of the bottom-up approach in a multidimensional protein identification technology platform (MudPIT), wherein a highly complex peptide mixture was first separated by two-dimensional cationexchange- and reversed-phase chromatography followed by online peptide identification using automated MS/MS and database searching.9 This approach has been successfully utilized to characterize the proteomes of several organisms and tissues, including yeast,20-22 bacteria,23,24 Caenorhabditis elegans,25 plants,26 mammalians,27 human serum,28,29 and tumor tissues30,31 An important element of multidimensional protein identification technology is the efficient separation of peptides after first-dimension fractionation and prior to mass spectrometric identification. Because of its high resolution and the applicability of mobile phases that are compatible with electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), ion-pair reversed-phase HPLC represents the chromatographic mode of choice for on-line or off-line interfacing with MS and MS/MS.32,33 Nonpolar, monolithic stationary phases, either based on silica gel or poly-(styrene-divinylbenzene) copolymer (PS-DVB), were shown to be highly suitable for peptide separations with high speed or high peak capacities.34-40 We have recently demonstrated that PS-DVB monolithic capillary columns can be combined to form a basically orthogonal two-dimensional peptide separation system by using ion-pair reversed-phase chromatography at high and low pH.41,42 Moreover, a direct comparison of monolithic with microparticular columns has shown that consistently 50-100% more peptides were identified by HPLC-MS/MS using the more efficient monolithic columns.43 Nevertheless, PS-DVB monoliths have so far not been implemented for proteome analysis in the more common two-dimensional separation schemes involving ion-exchange chromatography in the first dimension, most probably due to the lack of suitable trapping columns for on-line or off-line concentration and desalting. In due consequence, we developed in this study a twodimensional separation system that incorporated a microparticular, polymeric strong cation-exchange column in the first dimension and monolithic PS-DVB columns for preconcentration and separation of peptide mixtures in the second separation dimension. Peptides obtained from the soluble proteome of Myxococcus xanthus DK1622 were utilized not only to carefully characterize the analytical system in terms of orthogonality, peptide carryover, protein coverage, and reproducibility of protein identifications, but also to obtain information about expressed proteins and protein complexes that are
potentially involved in the synthesis of secondary metabolites. The system is shown to have potential for the detection of the components of large multimodular multienzyme systems and for the multidimensional analysis of whole proteomes.
2. Experimental Section Chemicals and Materials. Acetonitrile (E Chromasolv), 3-[(3-cholamidopropyl)-dimethylammonio]-1-propansulfonate (CHAPS), dithiothreitol (min. 99%), 2-mercaptoethanol (>98%), and urea (g99.5%) were purchased from Sigma-Aldrich (Steinheim, Germany); ammonium hydrogencarbonate (g99.5%), iodoacetic acid (analytical reagent grade g99.5%), magnesium sulfate, and trifluoroacetic acid (g99.5%) were from Fluka (Buchs, Switzerland); sodium dihydrogenphosphate-1-hydrate (analytical reagent grade), acetone (analytical reagent grade), and methanol (analytical reagent grade) were from Merck (Darmstadt, Germany); sodium dodecyl sulfate (SDS), thiourea, tris-hydroxymethylaminomethane (TRIS), dipotassium hydrogenphosphate, and potassium dihydrogenphosphate were from Roth chemicals (Karlsruhe, Germany), and sodium chloride (analytical reagent grade) was from Gru ¨ ssing GmbH (GmbH, Filsum, Germany). Bio-Rad dye concentrate was obtained from Bio-Rad (Munich, Germany). Water used in the experiments was purified using a Purelab Ultra system (Elga, Siershahn, Switzerland). Agar and casitone were purchased from Difco Laboratories (Michigan). For the preparation of growth media and plates, all substances were mixed and autoclaved. Trypsin (sequencing grade modified) was obtained from Promega (Madison, WI). Preparation and Digestion of the M. xanthus DK1622 Proteome. M. xanthus strain DK1622 was obtained from Dale Kaiser (Stanford University, Palo Alto, CA). M. xanthus was taken from a -80 °C stock culture and plated on CTT-agar plates (10 g L-1 casitone, 10 mL L-1 1 mol L-1 TRIS buffer (pH 8.0), 1 mL L-1 potassium hydrogenphosphate buffer (pH 7.6), and 10 mL L-1 0.8 mol L-1 magnesium sulfate; for agar plates, 15 g agar/L). Bacteria were grown at 30 °C in autoclaved Erlenmeyer flasks utilizing an incubator shaker (Infors, Einsbach, Germany) at 170 rpm. A single colony was taken to inoculate a 10 mL CTT preculture in a 50 mL Erlenmeyer flask. The preculture was incubated for 2 days; 500 µL of the wellgrown (OD600 ca. 3.3, undiluted) preculture was taken to start the 50 mL CTT main culture to an optical density (OD600nm) of 0.062 in a 250 mL Erlenmeyer flask. After 41.5 h of incubation, the culture was harvested at an OD600nm of 3.011 (average of 3 measurements, 1:3 diluted) by centrifugation (Beckman coulter Avanti JE, rotor JA14, 15 min. 10 000 rpm, 4 °C). The supernatant was removed, and the cells were frozen at -20 °C. Subsequently, the cell pellet was resuspended in lysis buffer (5 mol L-1 urea, 2 mol L-1 thiourea, 2% CHAPS, and 1% SDS), and cell lysis was performed by vortexing (vortex genie 2, Bender&Hobein AG, Zu¨rich, Switzerland) for 5 cycles (30 s each) using glass beads (425-600 µm, Sigma Aldrich) followed each time by 2-min incubations on ice. Cell debris was removed by centrifugation (Heraeus Christ, Minifuge T, Hanau, Germany) for 15 min at 4000 rpm and 4 °C. To remove nucleic acids, lipids, and salts, protein precipitation with acetone/methanol was applied.44 The mixture was kept at -20 °C for 3 h before the precipitated proteins were pelleted by centrifugation (5 min, 4000 rpm, 4 °C). The supernatant was removed carefully, and the remaining protein pellet was resolubilized in sample buffer containing 4 mol L-1 urea and 30 mmol L-1 TRIS (pH 6.5). The protein concentration was estimated using a Bradford protein Journal of Proteome Research • Vol. 5, No. 10, 2006 2761
research articles assay calibrated with bovine serum albumin.45 The protein extract was diluted by a factor of 10 before measurement. The dye reaction was incubated for 15 min at room temperature and measured in 96-well microtiterplates, using a plate reader (Bio-tek, Bad Friedrichshall, Germany) at 595 nm. The estimated protein concentration was 3 µg µL-1. Approximately 300 µg of the M. xanthus lysate was denatured in 160 µL of 8.0 mol L-1 aqueous urea/0.50 mol L-1 aqueous ammonium hydrogencarbonate solution for 30 min at 37 °C. Subsequently, the cystines were reduced with 60 µL of 300 mmol L-1 aqueous dithiothreitol solution for 4 h at 37 °C and carboxymethylated with 2.0 mol L-1 iodoacetic acid (4.0 µL) for 15 min at room temperature. The excess of iodoacetic acid was removed by addition of 8.0 µL of 2-mercaptoethanol (1.0 mol L-1). Dialysis of the mixture was performed in a 500 µL dialysis cassette with a molecular weight cutoff of 3500 Da (Slide-A-Lyzer, Perbio Science, Bonn, Germany) for 24 h against 1 L of distilled water. The dialyzed protein solution was subsequently digested with 20 µg of trypsin dissolved in 20 µL of trypsin resuspension buffer (activated for 30 min at 37 °C). Finally, the digestion was quenched after 12 h by addition of 1.0% (v/v) trifluoroacetic acid. Instrumentation. The chromatographic system for firstdimension ion-exchange HPLC consisted of a low-pressure gradient pump (Model M480G, Gynkotek, Germering, Germany), a 2-channel degasser (Model K-5002, Knauer, Berlin, Germany), a manual injector with a 200 µL sample loop (Model 7125, Rheodyne, Rohnert Park, CA), a polymer-based strong cation-exchange column (ProPac SCX-10, 250 × 4.0 mm, Dionex, Idstein, Germany), and a UV-detector with a 12-µL flow cell (Model 731.87, Knauer) set at 214 nm. A vacuum concentrator (model 5301, Eppendorf AG, Hamburg, Germany) was utilized to concentrate the collected fractions from cationexchange fractionation. The instrument utilized for second-dimension ion-pair reversed-phase HPLC consisted of a capillary/nanoHPLC system (Model Ultimate, LC Packings, Amsterdam, The Netherlands), an autoinjector (Model Famos, LC Packings), and a loading pump with a 10-port switching valve (Model Switchos, LC Packings). The preconcentration- (10 × 0.20 mm) and separation column (60 × 0.10 mm) contained a monolithic PSDVB-based stationary phase, prepared according to the published protocol46 (columns available from LC Packings-A Dionex Company). The preconcentration column and separation column were connected to the autosampler and 10-port valve so that the trapped analytes were transferred from the preconcentration column to the separation column in backflush mode. An ion trap mass spectrometer (model esquire HCT, Bruker Daltonics, Bremen, Germany) equipped with a modified ESIion source (spray capillary: fused silica capillary, 0.090 mm o.d., 0.020 mm i.d.) was utilized for mass spectrometric detection. MS/MS spectra were recorded in positive ion mode with an electrospray voltage of 3.5 kV and fragmentation amplitude ramped from 0.5 to 3.0 V. The heated capillary temperature was set to 300 °C. Mass spectrometric parameters for automated peptide identifications by data-dependent tandem mass spectrometry were as follows: mass range mode, ultra scan 50-3000 m/z; scan speed, 26.000 m/z per s, full scan, 400-1500 m/z; ion polarity, positive; trap drive, 93.2; octopole RF amplitude, 88.5 Vpp; lens 2, -36.1 V; capillary exit, 253.8 V; dry temperature, 300 °C; nebulizer, 20 psi; dry gas, 4 L min-1; high voltage capillary, -3500 V; high voltage end plate offset, 2762
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-500 V; ICC target, 70 000; maximum accumulation time, 200 ms; precursor ions auto MS(n), 3; MS averages, 5 spectra; MS/ MS scan range, 200-2000 m/z; active exclusion, after 2 spectra for 0.60 min; MS/MS fragmentation amplitude, 1.5 V; smart fragmentation, on (30-200%); absolute treshold MS/MS, 2000. Data Processing and Evaluation. The software DataAnalysis 3.1 from Bruker was used to evaluate the recorded MS data, using the following parameters for compound detection in chromatograms: S/N threshold, 0.001; relative area threshold, 0.1%; relative intensity threshold, 0.1%; skim ratio, 0.1; smoothing width, 1. Generation of the mass list was accomplished by the peak finder algorithm apex and the parameters: peak width (fwhm), 0.1 m/z; S/N threshold, 1; relative intensity threshold (base peak), 2%; absolute intensity threshold, 100. The settings for charge deconvolution were resolved-isotope deconvolution, maximum charge 3; related-ion deconvolution, maximum charge 4; MW agreement, 5 [0.01%]; minimum peaks in component, 3. Database searching was accomplished with Biotools software version 2.2 (Bruker, Bremen, Germany) and the Mascot software version 2.0 from Matrix Science (Matrix Science, London, U.K.), which is based on the MOWSE (Molecular Weight Search) algorithm.47,48 The acquired MS/MS spectra were searched against an in-house database containing the known open reading frames of M. xanthus DK 1622 (7413 entries49, http://www.tigr.org/tdb/mdb/mdbinprogress.html), using the following parameters: taxonomy, all entries; fixed modification, carboxymethylated; enzyme, trypsin; peptide tolerance, (1.3 Da; MS/MS tolerance, (0.3 Da; maximum number of missing cleavages, 2. Each protein was positively identified, if the hit was within a 95% significance level, meaning that a random hit occurred only with a frequency of less than 5%. The criterion for positive peptide identification was an ion score of greater than 27, because this value was computed by Mascot as threshold value for a significant hit for nearly all identified peptides. Isoelectric points (pI) of the identified peptides were predicted using the protein/peptide characterization tool ProtParam provided on the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (http://www.expasy.org/tools/protparam.html). Multidimensional Proteome Analysis of M. xanthus DK1622. One hundred microliters of the M. xanthus lysate was injected onto the strong cation-exchange column for first-dimension separation. Since a large portion of the tryptic peptides eluted during the early phase of the cation-exchange fractionation, a very shallow salt gradient was ramped in the first 9.0 min from 0 to 0.015 mol L-1 sodium chloride in 0.050 mol L-1 sodium dihydrogenphosphate buffer, pH 3.0, containing 20% acetonitrile, followed by 0.013-0.050 mol L-1 sodium chloride in 8.0 min, and finally 0.050-0.50 mol L-1 sodium chloride in 4.0 min. The column temperature was 25 °C. At a flow rate of 1.0 mL min-1, 0.50-min fractions were collected during the first 10 min of the fractionation, while 1.0-min fractions were taken for the rest of the chromatographic run. Each fraction was concentrated in a vacuum concentrator to a volume of 100 µL (5- and 10-fold concentration for the 0.50- and 1.0-mL fractions, respectively). A 10 µL aliquot of each concentrated fraction was injected into the second dimension for ion-pair reversed-phase separation. The fractions were desalted and concentrated for 4.0 min at a flow rate of 10 µL min-1 with 0.10% aqueous heptafluorobutyric acid solution on a short monolithic PS-DVB preconcentration column (10 × 0.20 mm i.d.) and subsequently transferred in backflush mode to
Proteome Analysis of Myxococcus xanthus
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Figure 1. Two-dimensional fractionation of a tryptic digest of a M. xanthus lysate by (a) first-dimension strong cation-exchange chromatography and (b-f) second-dimension ion-pair reversed-phase chromatography. Sample, (a) 100 µL of digest of M. xanthus lysate; fractionation, 18 0.5-min fractions (1-10 min), 18 1.0-min fractions (10-28 min); (b-f) 10 µL of SCX fractions nos. 1, 19, 25-27. Chromatographic conditions are given in the Experimental Section.
the 60 × 0.10 mm monolithic separation column. The peptides were eluted at 25 °C and a flow rate of 600 nL min-1 with a 90-min gradient of 0-20% acetonitrile in 0.050% aqueous trifluoroacetic acid. The tryptic peptides were on-line-detected by data-dependent ESI-MS/MS and subsequently identified by Mascot database search.
3. Results and Discussion Setup of a Two-Dimensional Separation System Using Monolithic Columns. The peak capacity even of the most efficient chromatographic separation techniques is usually insufficient to be able to adequately fractionate complex peptide mixtures resulting from the proteolysis of whole proteomes before mass spectrometric investigation. Hence, the combination of different separation stages in two-dimensional separation schemes is generally mandatory. There are two principal approaches to the two-dimensional chromatographic fractionation of highly complex peptide mixtures. In on-line systems, the individual separation dimensions are either realized by the combination of two separate separation columns that are connected by suitable switching valves50 or by two segments of two different stationary phases that are sequentially packed into a single column tube.9 This setup guarantees full automation and the compatibility with minute sample amounts due to the minimal loss of sample during the transfer from the first to the second dimension. However, flow rates and mobile phase compositions must be matched for both dimensions in this setup, and the separations in both dimensions cannot be independently performed. Off-line methods involve the collection of a finite number of fractions from the first dimension and subsequent manual or robotic transfer and separation in the second dimension.29 This approach facilitates easy and independent optimization of both separation stages, allows the loading of larger sample amounts in the first separation dimension, permits additional sample manipulations, such as preconcentration or rebuffering, between the dimensions, and offers the possibility of repeated injection of
fractions. Off-line two-dimensional schemes normally utilize linear gradients in the first dimension, allowing efficient peptide separation as well as monitoring of the separation on-line by UV-detection. Because of the inherent advantages mentioned above, we decided to integrate the monolithic columns into an off-line two-dimensional separation scheme. A nonporous, poly-(ethylstyrene-divinylbenzene)-based stationary phase coated with sulfonic acid functional groups on top of a layer of a hydrophilic polymer was utilized for the fractionation of peptides by strong cation-exchange chromatography (SCX). To minimize secondary solvophobic interactions of peptides with the stationary phase in the strong cation-exchange separation, 20% acetonitrile was added to the phosphate buffer, pH 3.0. A very shallow, two-phase gradient of 0-0.050 mol L-1 sodium chloride was applied to fractionate the peptides before a steep gradient was ramped up to 0.50 mol L-1 sodium chloride in order to elute the strongly retained analytes from the column. Because of the high complexity of the sample, individual peaks were hardly distinguishable in the chromatogram. However, the UV-trace shown in Figure 1 allowed an estimation of the relative concentration of peptides eluting form the column and a corresponding adjustment of the fractionation time. Accordingly, fractions of 0.5 and 1.0 mL were taken in the early and later phases of the fractionation, respectively. After concentration under vacuum, the SCX fractions were desalted and concentrated by injection onto a short, monolithic preconcentration column using 0.10% aqueous heptafluorobutyric acid as loading solvent before transfer and separation in a 60 × 0.10 mm monolithic capillary column. The separation was performed with an acetonitrile gradient in 0.050% trifluoroacetic acid, which was shown to offer better detectabilities for peptides than 0.050% heptafluorobutyric acid.33 Figure 1b-f show representative reconstructed total ion current chromatograms (filtered to represent the total ion current of full-scans only) of the fractions number 1, 19, and 25-27. Because of the different fractionation times, the maximum signal intensities Journal of Proteome Research • Vol. 5, No. 10, 2006 2763
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Figure 2. Peptide identifications in different strong cationexchange fractions. The sum of overall peptide hits represents the total number of peptide identifications delivered by Mascot from the four replicate analyses of all SCX fractions. The number of identified peptides per fraction was obtained by eliminating multiple identifications of the same peptide in the same fraction. Finally, the number different identified peptides was obtained by counting only those peptides that have not been identified in a previous SCX fraction.
in the reconstructed total ion current chromatograms could be kept in a narrow range of (1-6) × 106 counts. The orthogonality of both separation dimensions can be deduced from the homogeneous elution of peptides during the whole separation window of the ion-pair reversed-phase separation. To obtain data about the reproducibility of peptide identifications by data-dependent mass spectrometry, all fractions were analyzed in quadruplicate. Peptide Identifications in the Digest of M. xanthus. The acquired mass spectrometric data were subjected to automated data analysis using Mascot with the parameter settings described in the Experimental Section. Figure 2 collects the number of peptide identifications in the SCX fractions no. 1-30. During the late phase of column washing and regeneration, no peptides were detected (fractions no. 31-36). The gray bars in the graph represent the total number of all peptide hits obtained in the four replicate analyses of each fraction. Adding together the hits in all fractions, a total number of 5290 significant peptide identifications (Mascot score > 27) was obtained. After elimination of repeated identifications of a particular peptide in the four analyses of the same SCX fraction, the number of peptide identifications indicated by the white bars was extracted. It can be seen that about 35-45% of all hits were unique in an SCX fraction. Since the same peptide may be found in more than one SCX fraction, the number of different peptides identified is further reduced. Thus, the black bars in Figure 2 designate the number of nonredundant peptide hits in an SCX fraction that have not been identified in a previous fraction. The total number of unique peptides detected and identified in the M. xanthus lysate was 1312. Twentyseven percent of them were identified in only one analysis of all 144 analyses (36 fractions × 4 replicates), 15% in two, 12% in three, and 46% in four or more analyses (up to 25). On average, each peptide was identified 2.5 times in four replicate analyses for each SCX fraction. The number of successful peptide identifications shows a bimodal distribution with maxima in fractions 1 and 19 and a minimum in fraction 10. About 37% of all peptides were detected in the fractions taken during the first 6 min of the 2764
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Figure 3. Dependence of cation-exchange elution time on the number of basic functional groups in the peptides.
SCX fractionation (fraction 1-10), while the rest of the peptides were identified in the fractions taken from 6 to 27 min (fraction 11-31). A comparison of the isoelectric points predicted for the identified peptide sequences revealed that there is no obvious correlation between pI and elution position in the SCX chromatogram. An example for the retention behavior of a pair of peptides differing in a terminal arginine is illustrated in Figure 1b,c (gray traces) by the extracted ion chromatograms of the two peptides ALAEQFVQGLPLNSTSATGAVR/R (calculated pI of 6.05 and 9.64, respectively) in fractions nos. 1 and 19. Upon addition of arginine, the retention time of the peptide shifted from 1.5 to 11.0 min in the SCX separation. For 17 different pairs of peptides investigated, the addition of a basic amino acid entailed an average shift in SCX retention time by 8.4 min. Not unexpectedly, the addition of an arginine also resulted in increased retention of the peptides in ion-pair reversed-phase chromatography (from 66.7 to 76.9 min, see Figure 1b,c). A closer inspection of the sequences of the identified peptides revealed that most of the peptides identified in fraction 1-10 were typical, fully digested tryptic peptides containing no basic amino acids other than carboxyterminal arginine or lysine and a free amino-terminus (894 peptides in total). Given a pH of 3.0 of the eluent utilized for SCX fractionation, these peptides can be expected to be doubly charged in solution. Only few of the peptides (8 peptides) eluting in the early fractions were singly charged because they did not contain a lysine or arginine at the carboxy terminus. They were for the most part the carboxy-terminal peptides of the digested proteins. The peptides identified under the second maximum of the distribution mostly contained two basic amino acids and were predominantly tryptic peptides containing an additional histidine in their sequence or one missed tryptic cleavage site (958 peptides). In total, 132 peptides were found containing more than two basic amino acids (three basic amino acids, 116 peptides; four basic amino acids, 15 peptides; five basic amino acids, 1 peptide). Figure 3 illustrates the allocation of the number of basic functional groups, including aminoterminus, side chains of arginine, lysine, and histidine, in the peptides to the fraction numbers, which readily explains the bimodal distribution of peptide hits over the SCX fractions. One can see that the first fractions were dominated by peptides carrying two positive charges. After approximately 10 fractions,
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Proteome Analysis of Myxococcus xanthus Table 1. Distribution of Peptide Identifications over More than One Single SCX Fraction No. of peptide identifications fract. no.
fract. no. + 0
fract. no. + 1
fract. no. + 2
fract. no. + 3
fract. no. + 4
fract. no. + 5
fract. no. + 6
fract. no. + 7
fract. no. + 10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
225 50 57 92 27 15 10 8 4 1 6 10 20 10 5 21 22 58 62 63 70 83 86 90 62 34 32 45 39 5
170 24 17 22 10 6 3 3
130 23 16 20 9 6 3 3
112 10 8 10 3 2 1 1
29 6 1 5 2
9
2
1
1
3 7 8 8 4 14 12 26 24 21 16 7 5 10 4 4 4 2
3 7 5 3 3 12 12 26 24 21 16 7 5 10 4 4 4 2
1 6 4 5 4 7 6 6 3 2
2 2 1
1
4 6 6 2 6
2 1
5 5 5 2
1
1
average
the abundance of these peptides decreased rapidly as the abundance of the triply charged peptides began to increase, resulting in a minimum of successful peptide identifications in fractions 10-12 and a second maximum in fractions 1922. The last few SCX fractions contained mainly peptides with 3, 4, and even 5 basic amino acids in their sequence. The sequential elution of peptides according to increasing charge is a clear indicator that strong cation-exchange chromatography using the ProPac SCX-10 stationary phase and adding 20% acetonitrile to the mobile phase separates peptides primarily according to their charge. Peptide Crossover between SCX Fractions and Orthogonality of Two-Dimensional Separation. The peptide crossover between the individual SCX fractions was inspected in detail. Table 1 summarizes the total number of peptides found in each of the fractions as well as the portion of these peptides that could be also detected in subsequent fractions. On average, 32% and 28% of the identified peptides were also found in the first and second subsequent fraction, respectively. One can clearly see on the basis of Table 1 that, even with 20% organic modifier in the SCX eluents, it was not possible to avoid completely peptide crossover. The effect is more pronounced in the first part of the SCX fractions because of the very shallow gradient of sodium chloride applied to elute the peptides and the narrow collection intervals during the first 18 fractions (see Figure 1). Crossover is less prevalent in later fractions, in which the fractionation interval was longer and the salt gradient was steeper. Interestingly, some of the peptides were identified in two fractions that were separated by one or more fractions in which the peptide could not be found. Of the 1312 unique
fract. no. + 1 [%]
fract. no. + 2 [%]
75.6 48.0 29.8 23.9 37.0 40.0 30.0 37.5 0.0 0.0 50.0 70.0 40.0 80.0 80.0 66.7 54.5 44.8 38.7 33.3 22.9 8.4 5.8 11.1 6.5 11.8 12.5 4.4 0.0 0.0 32.1
57.8 46.0 28.1 21.7 33.3 40.0 30.0 37.5 0.0 0.0 50.0 70.0 25.0 30.0 60.0 57.1 54.5 44.8 38.7 33.3 22.9 8.4 5.8 11.1 6.5 11.8 12.5 4.4 0.0 0.0 28.0
peptides identified in our analysis, 4.2% were isolated by 1 fraction, 0.7% by 2 fractions, and 0.1% by 4 fractions. Such a distribution of peptides over isolated SCX fractions was also reported by Maynard et al.22 and was attributed to local basic regions, mixed modes of electrostatic and solvophobic interactions with the stationary phase, or peptide aggregates that affect peptide fractionation. The crossover of peptides is not so critical when only identification of proteins is the aim of the study, as long as the peptide is found in at least one of the SCX fractions. The problem becomes, however, more challenging in attempts to perform quantitative, especially absolute quantitative, analysis of proteins.51 In this case, quantitation accuracy may be severely hampered because of elution of peptides in more than one fraction due to different response factors when the peptide is eluting in different chemical environments. Figure 4 illustrates an example of a peptide identified in two SCX fractions which were separated by 4 fractions. The peptide TTVTDQGASHAVTGTNLTPEGQACIQK (hypothetical protein MXAN 4900) could be identified in fractions 1-5 and, addtionally, in fraction 10. Figure 4 shows the reconstructed total ion current chromatogram together with the extracted ion trace of the corresponding peptide for fractions 1 and 10. A falsepositive identification of this peptide in the different fractions can be practically excluded because of the relatively high ion scores and identification reproducibilities (fraction 1, ion score 41, identification in 3 of 4 replicates; fraction 10, ion score 42, identification in 2 of 4 replicates), and the highly similar retention times of the pepides. The identity of the peptides is Journal of Proteome Research • Vol. 5, No. 10, 2006 2765
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Figure 4. Example of peptide distribution over isolated SCX fractions for the peptide TTVTDQGASHAVTGTNLTPEGQACIQK by means of the extracted ion chromatogram and the corresponding MS/MS spectrum. Conditions as in Figure 2.
Figure 5. Orthogonality of SCX-HPLC × RP-HPLC for M. xanthus tryptic peptides.
further corroborated by similarity of the peptide fragment fingerprints shown in Figure 4. To graphically visualize the orthogonality of the twodimensional separation system using a pellicular, polymerbased cation-exchange stationary phase and hydrophobic, polymeric monoliths, retention times in both chromatographic modes are plotted for one of the four replicate analyses in Figure 5. The 1410 peptide hits are distributed practically over the whole separation space, proving that the two-dimensional setup is orthogonal to a high degree. These results corroborate previous reports on the orthogonality of SCX- and IP-RP chromatography using silica-based strong cation-exchangeand reversed-phase materials.42 As already discussed above, the low number of peptide hits between 5 and 7 min in the first dimension is due to the dependence of SCX elution time on total peptide charge. 2766
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Protein Identifications in the M. xanthus Lysate. Proteins were considered as positively identified if their matches had a probability of less than 5% of being a random hit, corresponding to a statistical significance level of 95%. Protein MOWSE scores can be deduced for Table 3 in the Supporting Information. The histogram of significant protein hits shown in Figure 6 clearly reflects the bimodal distribution already observed in the peptide identifications. In total, 4738 protein hits were obtained (Figure 6a), which narrow down to 631 unique proteins identifications upon elimination of replicate identifications and multiple peptides for the same protein (Figure 6b). This number of identified proteins corresponds to approximately 10% of all open reading frames (ORFs) contained in the M. xanthus database. A full list of the proteins annotated to ORFs of the M. xanthus genome is available as Supporting Information (Table 3). The different colors in the histogram and the pie chart also give information about the reproducibility of protein identifications in the replicate analyses. The average recovery of proteins in the four replicate runs performed for the individual fractions was 2.6, which is almost identical with the recovery of the peptides. Of the 631 proteins, 247 or 39% were found in all four replicates. The number of proteins only once identified is slightly lower (223 or 35%). In sum, about one-third of the proteins could be identified in all runs and one-third in only 1 of 4 replicates. These data demonstrate that, although protein identifications by twodimensional HPLC-ESI-MS/MS are quite reproducible, repetitive analyses help to significantly increase proteome coverage. It is also clear, on the other hand, that single analyses of a proteome bear a considerable risk of numerous proteins remaining unidentified although they may be present in concentrations principally detectable by HPLC-MS/MS. This is especially important in the search for proteins corresponding to annotated gene clusters, such as PKS and NRPS, for which the identification of a protein is a definite proof that the gene is expressed, while the absence of a gene product does not imperatively mean that the gene is silent.
research articles
Proteome Analysis of Myxococcus xanthus
Table 2. Identified Proteins Corresponding to Various Biosynthetic Gene Clusters in M. xanthus DK1622
Figure 6. Overall hits (a) and unique identifications (b) of M. xanthus proteins in the different SCX fractions. The inset in (b) illustrates the reproducibilities of protein identifications in four replicate analyses of each SCX fraction.
Identification of Polyketide Synthetases and Nonribosomal Peptide Synthetases. Nonribosomal peptides and polyketides represent an interesting class of natural products showing extreme stuctural diversity and possessing a broad spectrum of pharmacological activities. They are synthesized from simple building blocks such as amino- or carboxylic acids on multimodular enzyme complexes called polyketide synthetases (PKS) and nonribosomal peptide synthetases (NRPS).52 Knowledge of the complete genome of M. xanthus DK1622 and homology searches with known enzymes enabled the annotation of at least 18 gene clusters associated with PKS and NRPS. Analyzing PKS and NRPS both at the genomic and proteomic level is of special interest to evaluate and exploit the theoretical genetic capacity of bacteria like M. xanthus DK1622 for the production of novel metabolites. The experiments described here analyzed the protein profile of M. xanthus DK1622 in the late exponential growth phase (cells typically reached ODs of 4) and provide a deep insight into the metabolic situation of the bacterium. This strain was formerly not known as a producer of natural products at all, but recently, myxalamides, myxovirescins, myxochromides, DKxanthenes (P. Meiser, and H. B. Bode, unpublished results), and myxochelines (M. O. Altmeyer, P. Meiser, and R. Mu ¨ ller, unpublished results) have been detected after close inspection of the secondary metabolite profile under different conditions.5,53,54 Although these data already prove M. xanthus to be a multiproducer of secondary metabolites, they do not explain the presence of 18 different biosynthetic gene clusters in the genome of the strain.5 Table 2 lists the proteins that have been identified and assigned to the different annotated gene clusters associated with PKS and NRPS. Most interestingly, the data presented identify fragments of the PKS and NRPS proteins corresponding to the four biosynthetic genes of the compounds mentioned above. To our knowledge, this is the first unam-
protein
MXAN number
molecular mass [Da]
associated gene cluster
PKS NRPS PKS/NRPS PKS NRPS NRPS PKS PKS/NRPS NRPS + Thioesterase NRPS NRPS
4527 4403 4299 4292 4079 4000 3932 3779 3636 2796 1607
550.283 526.063 318.239 198.448 486.832 495228 330.338 1.551.772 1.301.994 170.024 599.646
myxalamid unknown 1 Dkxanthene Dkxanthene myxochromid unknown 2 myxovisescin unknown 3 unknown 4 unknown 5 unknown 6
biguous proof of expression of such genes in any natural product-producing bacterium. Even more intriguingly, the expression of six additional biosynthetic gene clusters of currently unknown function in M. xanthus could be shown (Table 2). In the literature, such biosynthetic gene clusters of hypothetical function are often called “silent” or “cryptic”, and there is some speculation whether they are functional and therefore responsible for the production of metabolites. Here, we clearly demonstrate that in M. xanthus at least six of the remaining 12 gene clusters are indeed expressed as the corresponding high-molecular proteins are unequivocally detected. The identification of the expressed gene products now forms the basis for searches for their biosynthetic products.
4. Conclusions Off-line two-dimensional chromatographic separations using a microparticular, polymer-based strong cation-exchange stationary phase and a polymeric, monolithic reversed-phase stationary phase are shown to be suitable for the proteome analysis of bacterial protein lysates by the shotgun approach. The method is useful for efficient orthogonal separation and detection of peptides from bacterial lysates. The proteins identified by this approach set the stage for further directed protein analysis to gain a deeper biological understanding of the proteins involved in the production of secondary metabolites, such as the PKS and NRPS expressed by M. xanthus DK1622. The advantage of this method is not only the identification of many expressed proteins, but also the possibility to detect proteins almost without size limit, which was difficult if not impossible in our attempts using two-dimensional gel electrophoresis, spot picking, and MALDI-MS/MS, especially if the expression of PKS and NRPS genes is to be investigated. The fact that several gene products for PKS and NRPS were detected strongly argues for a physiological role of their products, because an enormous amount of energy is obviously consumed to keep the large-size gene clusters in the chromosome and to express the corresponding biosynthetic proteins. For future studies, one can imagine to focus on the comparison of production spectra under different physiological conditions of M. xanthus wild-type cells and mutants generated in the described biosynthetic gene clusters. Similar approaches have already led to the identification of novel compounds in Stigmatella aurantiaca,4,55,56 but these studies were not guided by the pre-identification of expressed gene clusters which becomes possible due to the method described here. Our analysis already shows that the technique of 2D-HPLC-MS/ MS will be a powerful tool which not only provides deep insight Journal of Proteome Research • Vol. 5, No. 10, 2006 2767
research articles into the highly complex mixture of proteins in cells at a certain point of cultivation, but also can provide a detailed overview of the expression patterns of large PKS and NRPS proteins.
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PR0602489
