Quantification of the Proteins of the Bacterial Ribosome Using

Article pubs.acs.org/jpr

Quantification of the Proteins of the Bacterial Ribosome Using QconCAT Technology Zubida M. Al-Majdoub,†,‡ Kathleen M. Carroll,† Simon J. Gaskell,§ and Jill Barber*,†,‡ †

Manchester Institute for Biotechnology, 131 Princess Street, Manchester M1 7DS, United Kingdom Manchester Pharmacy School, University of Manchester, Manchester M13 9PT, United Kingdom § Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom ‡

S Supporting Information *

ABSTRACT: The bacterial ribosome is a complex of three strands of RNA and approximately 55 proteins. During protein synthesis, the ribosome interacts with other proteins, numbered in the hundreds, forming some stable and some transient complexes. The stoichiometries of these complexes and of partially assembled ribosomes are often unknown. We describe the development of a flexible standard for the determination of stoichiometries of ribosomal particles and complexes. A core QconCAT, an artificial protein consisting of concatenated signature peptides derived from the ribosomal proteins L2, L4, L13, S4, S7, and S8, was developed. The core QconCAT DNA construct incorporates restriction sites for the insertion of cassettes encoding signature peptides from additional proteins under study. Two cassettes encoding signature peptides from the remaining 30S and 50S ribosomal proteins were prepared, and the resulting QconCATs were expressed, digested, and analyzed by mass spectrometry. The majority of Escherichia coli ribosomal proteins are small and basic; therefore, tryptic digestion alone yields insufficient signature peptides for quantification of all of the proteins. The ribosomal QconCATs therefore rely on a dual-enzyme strategy: endoproteinase LysC digestion and analysis followed by trypsin digestion and further analysis. The utility of technology was demonstrated by a determination of the effect of gentamicin on the protein composition of the E. coli ribosome. KEYWORDS: Bacterial ribosome, ribosome complexes, stoichiometry, signature peptides

■

INTRODUCTION The bacterial ribosome is a complex structure consisting of three RNA molecules and about 55 proteins arranged in two distinct subunits. It is the central machinery in bacterial protein synthesis and is known to interact with more than 100 other proteins during protein synthesis.1 Because of its central importance in cellular function, the ribosome is the target for seven classes of antibacterial agents, including the aminoglycosides, arguably the most effective antibacterial agents in the clinic, and the macrolides, probably the safest antibacterial drugs.2 Elegant crystal structures of prokaryotic ribosomes and subunits were rewarded with the 2009 Nobel Prize for Chemistry.3−5 Key to understanding the interactions of the ribosome with other proteins is the question of stoichiometry. How many molecules of these factors bind to one ribosome? Stoichiometry is also key to understanding ribosomal assembly and especially the effects of drugs, including the macrolides and aminoglycosides, on this process.6,7 There are many powerful, sensitive methods for relative (between samples) quantification of proteins based on mass spectrometry, with the SILAC and iTRAQ methods being especially widely applicable.8,9 For stoichiometric (within sample) measurements, however, an absolute quantification method is required. The choices here are © 2014 American Chemical Society

quite limited: quantification based on standard peptides labeled with stable isotopes10 is attractive when single measurements are to be made, but when the stoichiometries of the same proteins are to be measured repeatedly, QconCAT methodology has many advantages.11 A QconCAT is an artificial protein expressed using a gene construct in Escherichia coli. The protein consists of signature peptides from all of the proteins to be quantified, which are concatenated. When a known amount of stable isotope-labeled QconCAT is digested with an unlabeled sample, the signature peptides appear as doublets in the mass spectrum; thus, unlabeled signature peptides from the sample may be quantified by comparison with their isotope-labeled counterparts derived from the QconCAT. Our aim was to prepare a flexible QconCAT to use for the determination of ribosomal stoichiometries and the stoichiometries of factors bound to bacterial ribosomes. Received: July 1, 2013 Published: February 4, 2014 1211

dx.doi.org/10.1021/pr400667h | J. Proteome Res. 2014, 13, 1211−1222

Journal of Proteome Research

■

Article

streaked onto an LB agar plate containing ampicillin at 50 μg mL−1 and incubated at 37 °C overnight.

EXPERIMENTAL SECTION

Materials and Reagents

Protein Expression

Trypsin (sequencing grade) was purchased from Roche Diagnostics, lysyl endoproteniase (Lys-C) was obtained from Wako (Osaka, Japan), and RapiGest was obtained from Waters (Bedford, MA, USA). All other chemicals and solvents (HPLC grade) were purchased from Sigma-Aldrich (UK). [13C6]K and [13C6]R and an unlabeled standard peptide (GGVNDNEEGFFSAR) used as an internal standard were purchased from Cambridge Isotope Laboratories (UK).

A single colony from the plate was used to inoculate 5 mL of LB medium containing 50 μg mL−1 ampicillin, and this was incubated overnight at 37 °C with shaking at 220 rpm. The overnight culture was then added to 250 mL of LB medium in a 1 L flask. This was incubated with shaking (220 rpm) until the OD600 achieved 0.6−0.8. Protein expression from the plasmid was then induced by addition of isopropyl-Dthiogalactoside (IPTG) to a final concentration of 500 μM. Incubation continued for a further 4 h unless otherwise stated. Samples were collected at 0, 1, 2, 3, and 4 h after induction for SDS-PAGE analyses. Last, cells were collected by centrifugation at 4200g for 10 min at 4 °C.

In Silico Digestion of 70S Ribosomal Proteins

In silico digestion of E. coli ribosomal proteins was carried out using Protein Prospector.12 The ribosomal proteins were digested with trypsin, endoproteinase Lys-C, endoproteinase Asp-N, and chymotrypsin alone as well as in combination. Sequential digestion with endoproteinase Lys-C and trypsin was found to yield the greatest number of proteotypic signature peptides for the bacterial ribosomal proteins. This Lys-C/trypsin sequential-digestion strategy was adopted in this study (for further detail, see Supporting Information Table S1).

Protein Expression of Stable Isotope-Labeled Proteins

Transformed BL21 cells were grown in M9 minimal medium (supplemented with 20% glucose, 1 M MgSO4, 0.1 M CaCl2, and 0.5% thiamine) in the presence of ampicillin (50 μg mL−1). A full complement of amino acids was added. All amino acids other than arginine and lysine were suspended in sterile water at 10 mg mL−1 and added to the medium to a final concentration of 0.1 mg mL−1. The labeled amino acids ([13C6]R and [13C6]K) were weighed out separately, and the solids were added to the medium to a final concentration of 0.1 mg mL−1. An overnight starter culture was prepared by inoculating a 1 μL glycerol stock into 5 mL of M9 medium without amino acids and growing for 18 h at 37 °C. The overnight culture was added to 250 mL of M9 medium containing all of the amino acids including labeled arginine and lysine to give a starting OD600 of approximately 0.06−0.1. The cells were grown to an OD600 of 0.6−0.8 at 37 °C, after which isopropyl-D-thiogalactopyranoside (IPTG) was added to a final concentration of 500 μM, and cells were incubated for another 5 h. Samples were collected at 0, 1, 2, 3, 4, and 5 h of induction for SDS-PAGE analysis. Cells were harvested by centrifugation at 4200g for 10 min at 4 °C.

QconCAT Gene Design and Construction

Core, 30S, and 50S QconCAT peptide selection was based on experimental criteria; all peptides were detected by LC−MS/MS of digests of pure ribosomes. Two tryptic or Lys-C peptides (or one of each) were chosen to represent each ribosomal protein. The peptide sequences were then concatenated in silico and used to direct the design of a codon-optimized gene for expression in E. coli. The predicted transcript was analyzed for RNA secondary structure that might diminish expression. Additional peptide sequences were added to provide an initiator methionine (MGTK) and sacrificial peptides (LEPGR and KLPWR) for the core QconCAT (see Figures S1 and S2 in the Supporting Information): LEK, PGR, KLK, and PWR for 30S QconCAT (Figure 1) and LEK, APGK, KLR, and APWR for 50S QconCAT (Figure S3). A His6 sequence (LAAALEHHHHHH) was added for affinity purification. The full QconCAT sequences are shown in Figure 1 (30S QconCAT) and Supporting Information Figures S2 and S3 (core and 50S QconCATs). The artificial gene was designed from the chosen peptides, synthesized de novo, verified by DNA sequencing, and ligated into the NcoI, XhoI, SmaI, HindIII, and BamHI sites of the pET21a expression vector by Entelechon (http://www.qconcat.com/) to yield the QconCAT plasmids.

Cell Lysis

This procedure was adapted from Beynon et al. with minor modification.11 Pelleted cells were resuspended in Bugbuster protein extraction reagent (Merck); 6 mL of reagent was added to 1.19 g of wet cells, and the suspension was incubated at room temperature for 20 min with gentle agitation. Twenty microliters of the suspension (total fraction) was removed for analysis by SDS-PAGE. The remaining suspension was spun at 16 000g for 20 min at 4 °C. The supernatant (soluble fraction) was collected in a clean falcon tube, and 20 μL was removed for analysis by SDS-PAGE. The pellets were resuspended again with Bugbuster at the same ratio as before. Then, lysozyme (10 mg mL−1) was added with shaking (100 μL per gram of wet cells), and the suspension was left to stand for 5 min at room temperature. Twenty milliliters of diluted (1:10 in sterile water) Bugbuster was added to the suspension, which was gently shaken for 1 min, and the mixture was centrifuged at 16 000g for 15 min at 4 °C. Pellets (containing inclusion bodies) were washed three times with a diluted solution of Bugbuster (1:10 v/v in water, 20 mL), and centrifugation at 16 000g for 15 min at 4 °C was performed to repellet the inclusion bodies after each wash. Last, the pellets were solubilized in binding buffer (20 mM sodium phosphate buffer, pH 7.4, 20 mM imidazole, 0.5 M NaCl, and 8 M urea) with gentle shaking, and the suspension was centrifuged at 5000g for 5 min at room temperature to remove debris.

Preparation of Recombinant Proteins

The DNA constructs for the QconCATs were produced by PolyQuant GmbH (http://www.polyquant.com/) (Germany) using the expression vector pET21a encoding ampicillin and kanamycin resistance. The received plasmid (5−10 μg) was dissolved in 50 μL of distilled water, producing a final DNA concentration of approximately 100−200 ng μL−1. Transformation of QconCATs

E. coli BL21 (λDE3) competent cells (Novagen, UK) were used for QconCAT plasmid transformation. Ten microliters of BL21 competent bacteria was placed in a microcentrifuge tube and thawed on ice, and 0.5 ng of plasmid DNA was added. After incubation on ice for 10 min, cells were heat shocked at 42 °C for 30 s and then cooled on ice for 2 min. Then, 150 μL of Luria− Bertani (LB) medium containing 50 μg mL−1 of ampicillin was added, and the cells were incubated at 37 °C for 30 min with shaking. To select transformed bacteria, cells (100 μL) were 1212



Article

quadrupole mass spectrometer operating in positive ion mode using a nanoflow probe. Capillary voltages of 1.2−1.5 kV and cone voltages of 60−100 V were typically used. MaxEnt 1 processing was used to survey the spectra initially and to provide candidate species; reported m/z values for assigned proteins were calculated by conventional deconvolution of the chargestate distributions (Figure S4 in the Supporting Information).

Qualitative analysis by SDS-PAGE indicated that the core QconCAT was fractionated to high purity at this point. 30S and 50S QconCATs expressed in LB and minimal medium were further purified as outlined below. Purification and Analysis of QconCAT Proteins

The purification of core, 30S, and 50S QconCAT proteins from the solution obtained above was carried out using a 1 mL HisTrap column (GE Healthcare). The column was washed with 10 mL of water and then equilibrated with 25 mL of binding buffer using a flow rate at 0.5 mL min−1. The supernatant containing QconCAT protein was applied to the column at flow rate of 0.25 mL min−1. Nonspecifically bound protein was washed off using 25 mL of binding buffer at flow rate of 0.5 mL min−1. The bound protein was eluted from the column using 5 mL of elution buffer (20 mM sodium phosphate, pH 7.4, 500 mM imidazole, 0.5 M NaCl, and 8 M urea) at a flow rate of 0.25 mL min−1, collecting five 1 mL fractions. Proteins from each fraction were adsorbed onto StrataClean bead resin (Stratagene), and each suspension was analyzed by SDS-PAGE. The eluent fractions containing His6-tagged protein were dialyzed against 100 volumes of 10 mM ammonium bicarbonate at 4 °C for 2 h and then against fresh buffer overnight. The QconCAT solution was concentrated by ultrafiltration using a Vivaspin centrifugal concentrator with a 20 000 molecular weight cutoff (Sartorius, UK). The purified protein (4 mL) was centrifuged in the concentrator at 4000 rpm at 4 °C for 30 min. Typically, the protein volume would decrease by one-half upon spinning.

Digestion Protocols

Lys-C and Tryptic Digestions. QconCAT proteins (50 μg) were resuspended in 50 mL of ammonium bicarbonate 50 mM, pH 8.0, prior to digestion. Proteins were reduced with 5 mM DTT (final concentration) at 60 °C for 30 min. After cooling at room temperature, proteins were alkylated with 20 mM (final concentration) iodoacetamide, and the mixture was incubated for 45 min in the dark at room temperature. Lys-C or trypsin was added at a ratio of 1:50 (w/w), and the enzymatic digestion was allowed to proceed at 30 °C (Lys-C) or 37 °C (trypsin) for 18 h. The digestions were stopped by acidifying to pH 4 with TFA (1% solution), and the samples were loaded onto C18 ZipTips for desalting and concentration prior to MALDI-TOF MS analysis. Digestion Using RapiGest SF. This protocol was applied to core and 30S QconCAT and also to 70S ribosomal proteins samples. The QconCATs (50 μg) were solubilized in 0.1% (w/v) RapiGest SF (Waters, UK) solution prepared in 100 mM Tris, pH 8.0, buffer containing 2 mM CaCl2 (total volume 100 μL), reduced by the addition of DDT (5 mM final concentration) for 30 min at 50 °C, and alkylated by incubation with iodoacetamide (final concentration 15 mM) in the dark at room temperature for 30 min. Ten microliters of endoproteinase Lys-C (0.1 μg μL−1) was added, and the sample was incubated at 30 °C with shaking for 4 h. For complete digestion, a further aliquot of Lys-C was added, and the sample was incubated at 30 °C for 15−18 h with shaking. Where further digestion with trypsin was required, trypsin (1 μg) was added, and the protein was digested at 37 °C with shaking for 4 h; a further aliquot of trypsin was then added, and digestion was completed overnight. HCl irreversibly inactivates trypsin13 and also degrades the RapiGest surfactant. Following digestion, the RapiGest was precipitated by addition of 250 mM HCl (final concentration) and then removed as a cloudy pellet by centrifugation in a microfuge at 14 000 rpm for 10 min at 4 °C. The supernatant was removed and analyzed by MALDI-TOF MS or LC−MS/MS depending on the complexity of the samples. Digestion Using Urea Denaturation and Overnight Digestion. QconCAT samples (20 μL) were digested in the presence of a urea solution (6 M urea/2 M thiourea), as described elsewhere, with minor modifications.14 The proteins were evaporated to 10 μL, resuspended in 40 μL of urea solution (in 10 mM Hepes, pH 8.0), and incubated at room temperature for 15 min. Proteins were reduced by addition of 5 μL of 10 mM DTT (in 50 mM ammonium bicarbonate, pH 8.0) and incubated for 30 min at room temperature. Alkylation of cysteine residues was subsequently performed by addition of 5.5 μL of 55 mM iodoacetamide (in 50 mM ammonium bicarbonate, pH 8.0) and incubation for 20 min in the dark. After reduction and alkylation, endoproteinase Lys-C was added in ratio of 1:50 enzyme/protein followed by incubation for 4 h at 30 °C. To ensure complete proteolytic hydrolysis for peptide quantification, another aliquot of Lys-C was added after 4 h incubation, and the digest was allowed to continue at 30 °C for 18 h. Before tryptic digestion, the resulting peptide mixtures were diluted with 4 volumes of 50

Determination of QconCAT Concentration Using Bradford Assay

The concentration of QconCAT protein after purification was measured using the Bradford protein assay. The concentrations of labeled core, 30S, and 50S QconCAT were determined to be 1.8, 0.23, and 0.03 mg mL−1, respectively, in 2 mL of solution. Determination of 30S and 50S QconCAT Concentrations Using Standard Standard Peptide

The unlabeled peptide, GGVNDNEEGFFSAR, was stored lyophilized at −20 °C in a vial containing 0.7 mg of peptide. The peptide was thawed and reconstituted in water to generate a working solution of 1 μg μL−1. Briefly, 700 μL of water was added to the peptide vial. The vial was carefully mixed on a vortex mixer to dissolve the peptide fully. The stock solution was further diluted to generate a working solution of 0.5 pmole μL−1. For quantification of QconCAT, the standard peptide was added to digested labeled material in serial dilutions, and every dilution was analyzed in triplicate. Extracted ions chromatograms were taken for the monoisotopic peak of the quantification peptide GGVNDNEEGFFSAR derived from the QconCAT protein. The absolute concentration of the [13C6]R/K-labeled 30S QconCAT was determined by a serial dilution of the standard peptide, which was then added to the labeled QconCAT that had been digested with trypsin. This peptide mixture was then analyzed by MALDI-TOF MS and Q-TOF LC−MS. Each dilution was carried out in triplicate. The L/H ratios were then determined and used to quantify the amount of peptide released from the protein during incubation with trypsin at 37 °C. Electrospray Mass Spectrometry of Intact QconCATs

QconCATs were prepared at concentrations of 60 fmole μL−1 in 50% acetonitrile containing 0.1% formic acid immediately before electrospray ionization mass spectrometric analysis. Nanoflow capillaries were used to introduce the samples into a Micromass 1213



Article

(Thermo), composed of a 3500 kDa membrane, against a 10 mM ammonium carbonate solution. This was incubated with magnetic stirring for 2 h at 4 °C. The buffer was then removed and replaced with fresh buffer and incubated at 4 °C for 18 h.

mM ammonium bicarbonate, pH 7.9. Trypsin was added, and proteolysis was continued at 37 °C for 4 h. To this was added another aliquot of trypsin, and the digestion was allowed to continue at 37 °C for 18 h. Trypsin activity was quenched by acidification using TFA or formic acid to a final concentration of 1%. Each sample was then concentrated by centrifugal evaporation to a total volume of 20 μL. Samples were desalted using C18 ZipTips (Millipore, Watford, UK), and the eluted peptides were concentrated by centrifugal evaporation and dilution in 2% ACN and 0.1% formic acid or 0.1% trifluoroacetic acid (TFA) and then analyzed by MALDI-TOF and RP-LC− MS/MS, respectively.

Estimation of Ribosome Concentration at 260/280 nm

The concentration and purity of isolated 70S ribosomal proteins were determined by measuring the optical density (OD) at 260 and 280 nm in a Eppendorf Biophotometer (Helena Bioscience). An OD260 of 1 represents 24 pmole mL−1 of RNA.15 An OD260/ OD280 ratio between 1.8 and 2 indicates pure ribosomes. Contaminants that absorb at 280 nm (e.g., protein) will lower this ratio. The 70S ribosomal protein samples from both control and treated samples gave ratio OD260/280 = 1.6−2.0, as expected for this crude ribosome preparation. The 70S ribosome samples were then aliquoted and stored at −80 °C until further use.

Guanidination

Peptides obtained by Lys-C digestion were treated with 7 M ammonium hydroxide (10 μL) and 0.5 M O-methylisourea (6 μL) for 65 °C for 12 min. TFA (1%, 3 μL) was then added to stop the reaction. The samples were desalted using C18 ZipTips before MALDI-TOF analysis. The conversion of lysine to homoarginine by guanidination increases the basicity of lysine-terminated peptides and improves their response factors in the mass spectrometer.

In-Solution Digestion of a Mixture of Unlabeled 70S Ribosomes and Labeled 30S QconCAT

Isolated 70S ribosomes (52.2 μg) were spiked with 300 and 600 ng of labeled 30S QconCAT for tryptic and Lys-C digests, respectively, and the mixtures (both control and gentamicintreated samples) were evaporated to 10 μL and then resuspended in 40 μL of denaturing solution (6 M urea/2 M thiourea). Digestion with Lys-C and trypsin was carried out using the procedure for digestion in the presence of urea/thiourea as described above.

MALDI-TOF Mass Spectrometry Analysis

Experiments were performed on an Ultraflex II TOF/TOF mass spectrometer (Bruker Daltonics, Germany) equipped with a nitrogen UV laser (337 nm) under the control of the FlexControl software. MS spectra were analyzed with FlexAnalysis 2.2 software. The TOF spectra were recorded in the positive ion reflector mode over a mass range of 700−3000 m/z. The ion acceleration voltage used was 25 kV. A solution of α-cyano-4hydroxycinnamic acid in 70% (v/v) acetonitrile and 0.1% (v/v) TFA at a concentration of 10 mg mL−1 was used as the matrix.

LC−MS/MS Operating Conditions

The Lys-C and tryptic peptide mixtures were separated by LC− MS using a nanoACQUITY chromatograph (Waters MS Technologies, Manchester, UK) coupled to an LTQ Orbitrap mass spectrometer XL (ThermoFisher Scientific, Bremen, Germany) with the manufacturer’s dynamic nanospray source fitted with a coated PicoTip Emitter 20−10 μm (New Objective, MA, USA) and with the voltage applied at the tip. The sample temperature was maintained at 10 °C, and 4 μL of each peptide sample was injected initially onto a trapping column (C18, 180 μm × 20 mm, Waters MS Technologies, Manchester, UK) using the partial loop mode of injection at a flow rate of 18 μL min−1. Chromatographic separation was performed on a reversed-phase C18 analytical column (nanoACQUITY UPLC BEH C18 75 μm × 150 mm 1.7 μm column), and the column temperature was set at 35 °C and developed at 300 nL min−1. The aqueous mobile phase (A) consisted of HPLC-grade water with 1% (v/v) formic acid; the organic phase (B) was 100% acetonitrile with 1% (v/v) formic acid. The organic composition was increased gradually from 1 (v/v) to 50% (v/v) buffer B over 30 min followed by a rapid ramp to 85% buffer B over 1 min and then a return to the initial conditions for re-equilibration prior to the next injection.

Isolation of Total Ribosomal Proteins

The strain used in this study was E. coli K-12 (strain KS1000, New England Biolabs, UK). The cells were grown at 37 °C in 200 mL of LB with aeration until the OD600 reached 0.2. At this point, gentamicin (final concentration, 6 μg mL−1) was added to three cultures; no antibiotic was added to the control cultures. All experiments were carried out in triplicate. Cells were harvested at midlog growth phase (OD600 of ∼0.8 for control and ∼0.6 for treated cultures) by centrifugation at 4200g for 15 min, and the pellets washed twice in ice-cold phosphate buffered saline (PBS), pH 7.4. Cells were resuspended in sterile ribosome lysis buffer (RLB) containing tris(hydroxymethyl)-aminomethane (Tris) buffer, 20 mM, pH 7.5, 50 mM magnesium acetate, 100 mM ammonium chloride, 1.0 mM DDT, and 0.5 mM ethylenediamine tetraacetic acid (EDTA). The cells were sonically treated: 10 repetitions of a 15 s burst at 35% power followed by a 45 s pause to keep the extract cool. Deoxyribonuclease was added directly to the lysate at a final concentration of 2 μg mL−1 to degrade contaminant DNA, and the mixture was incubated for 20 min at 4 °C. The mixture from the lysed cells was centrifuged at 30 000g for 30 min at 4 °C to remove debris and unbroken cells. This step was repeated twice. To separate 70S ribosomes from the remaining cellular components, the clarified supernatant was layered over a 1.1 M sucrose solution in ribosome lysis buffer (RLB), and the 70S ribosomes were pelleted by spinning at 100 000g at 4 °C in a Ti 50.2 rotor (Beckman Coulter, Fullerton, CA) for 16 h. The ribosomal pellet was resuspended in a small volume (3 mL) of RLB. Salts and low-molecular mass components were removed from the ribosome samples using a Slide-A-Lyzer dialysis cassette

Mass Spectrometry

The LTQ Orbitrap XL was calibrated prior to use according to the manufacturer’s instructions, and the data were acquired using Xcalibur version 2.0.5/Tuneplus version 2.4SP1/configured with Waters Acquity driver (build 1.0) as described.11 For quantification applications, full-scan MS spectra (m/z range, 300−1600) were acquired with the Orbitrap operating at a resolution (R) of 30 000 (as defined at m/z 400) and alternated with MS/MS scans. Data were acquired with a preferred inclusion list (containing both 30S Lys-C and tryptic peptides, heavy and light analogues), such that either the most intense precursor from m/z list was selected or the most intense ion from the MS1 scan was selected if no listed precursor was detected. This approach was used to maximize the data points across the 1214



Article

for some peptides in which Pride Wizard failed to measure theses peptides (Table 4). Absolute quantification is obtained by multiplying this ratio by the known amount of the standard. Raw data are appended in the Supporting Information.

chromatographic peak while concomitantly acquiring tandem MS data for sequence verification. A normalized collision energy of 30% was applied with an activation q of 0.25, and helium was used as the collision gas. Dynamic exclusion was enabled for 30 s with a repeat count of two for a duration time of 180 s, and all product ion spectra were acquired in the LTQ (ITMS m/z isolation width = 2). The automatic gain control (AGC) feature was used to control the number of ions in the linear trap and was set to 1 × 106 charges for a full MS scan and 1 × 104 for the LTQ (MSn), with max injection times of 50 ms applied for the LTQ. Sample acquisitions were alternated with buffer-only blank (defined as the starting mobile phase) injections to ensure data analysis/ quantification was not compromised by sample carryover. Data analysis was carried out using the Qual Browser (version 2) component of Xcalibur 2.0.6.

■

RESULTS

Core QconCAT

We required a strategy that would enable us to measure the stoichiometries of ribosomal proteins and other factors (especially protein synthesis factors) relative to one another and relative to ribosomes. The sheer number of proteins to be assessed makes a single QconCAT impractical. The approach we adopted was to develop a core QconCAT consisting of signature peptides derived from six ribosomal proteins, L2, L4, L13, S4, S7, and S8, located in the core of their respective subunits. These proteins were chosen because they are assembled early and are believed to be essential for ribosomal function. Signature peptides were identified experimentally by mass spectrometric analyses of digested ribosomal proteins using a variety of methods based on both MALDI MS/MS and ESI LC−MS/ MS.19 An ideal signature peptide must show a high signal response in the mass spectrometer, a low susceptibility to modification (such as methionine oxidation) or missed cleavage, and no post-translational modification (PTM) listed in the SwissProt database to minimize the presence of variant forms. Because the ribosomal QconCATs are intended for use by groups outside the specialist mass spectrometry community, simple detection by ESI and/or MALDI mass spectrometry is highly desirable. The process of identification of signature peptides uncovered an immediate problem, which was confirmed by in silico digestion of all of the E. coli ribosomal proteins. Most ribosomal proteins are small and basic, and many, including L13 and S7, give fewer than the preferred two proteotypic peptides on tryptic digest, mainly because of missed cleavage in regions with several basic residues. In silico digestion with other readily available single proteolytic enzymes gave no improvement but suggested the following strategy: Lys-C digestion, analysis, tryptic digestion, and analysis. The digestion with endoproteinase LysC before tryptic digestion (reminiscent of the MudPIT experiment)20 not only results in additional peptides available for analysis but also directs the cleavage to lysine in regions rich in basic residues, resulting in reduced missed cleavage. A summary of these results is shown in Supporting Information Table S1. The sequence of the core QconCAT and its gene are shown in Figure S1. In addition to the ribosomal peptides, the construct contains two sacrificial peptides that, at the DNA level, contain restriction sites for the insertion of cassettes encoding groups of signature peptides. It also contains two QCAL peptides21 for very accurate absolute quantification of the QconCAT and a His6-tag for purification (Table S2). Endoproteinase Lys-C is generally believed to cleave at KP motifs, but two of the chosen signature peptides contain a KPE motif, and there is no evidence of these being substrates for the enzyme (Table S2). The core QconCAT was expressed in E. coli in inclusion bodies (Figure S2). Isolation from washed inclusion bodies yielded QconCAT that was pure by SDS-PAGE, and affinity purification using the His6-tag afforded no advantage. Digestion using endoproteinase Lys-C yielded the expected peptides with no evidence of missed cleavage (Figure S2). A C-terminal peptide at m/z 1961.9 contains the His6-tag and part of the sacrificial

Raw LC−MS/MS Data Analysis

Fragmented Lys-C and tryptic peptides were identified using the Mascot (version 2.1.0) software platform (Matrix Science, London, UK). LTQ Orbitrap spectra from the 30S QconCAT and isolated ribosomal protein were searched against the E. coli K12 database with taxonomy: E. coli (21.10.10, 4339 sequences), trypsin or Lys-C with a maximum two missed cleavages, and carbamidomethyl (C) as fixed modification, whereas labeled [13C6]K and [13C6]R and oxidized methionine were searched as variable modifications. Peptide tolerance was set to 50 ppm with 2+ and 3+ peptide charges and 0.8 Da for CID fragment ions. For confident identification, peptides were identified using both MS and MS/MS. A peptide rank of 1 was required. Because of the high mass accuracy, the 95% significance threshold in the E. coli K12 database search was a Mascot score of 20. Absolute Quantification Using QconCAT PrideWizard

Automated quantification of peptide pairs from ribosomal proteins and the 30S QconCAT was performed using QconCAT Pride Wizard, an extension of the original Pride Wizard, which was developed to quantify iTRAQ-labeled samples.16,17 The Pride Wizard provides a user interface to which batches of spectra may be submitted. 30S QconCAT-labeled peptides are then identified through a Mascot MS/MS ion search. Protein hits are filtered such that only those that contain at least one QconCATlabeled peptide with a rank of 1 and a peptide expect score < 5 are retained. Furthermore, peptides are filtered such that the only ones quantified are unmodified (apart from the QconCAT label) and are unique to a single protein. In this study, analysis was performed on all replicates (a group of 36 Lys-C and trypsin spectra were analyzed using the QconCAT Pride Wizard). Quantification was then performed by first generating an extracted ion chromatogram for the m/z value corresponding to the precursor ion matching each labeled peptide. Where multiple matches occur against the same labeled peptide in a given protein, the highest scoring one is considered for quantification. Savitzky−Golay smoothing18 is applied to the chromatogram, and the starting and ending retention times for the chromatographic peak matching the peptide are determined on the basis of the retention time of the fragmentation spectrum that supplied the peptide match. Each precursor scan within this retention-time window is extracted and analyzed with an implementation of the SILAC analyzer linear-fit quantification algorithm. This provides a light/heavy ratio and standard error for each identified unlabeled/labeled peptide pair (Table 4). Ratios of unlabeled to labeled peptide areas were calculated and averaged across replicates. Manual analysis was also performed 1215



Article

Figure 1. 30S ribosomal QconCAT (a) sequence showing Lys-C peptides in blue, tryptic peptides in green, and sacrificial peptides in orange. KP sequences are underlined. (b) SDS-PAGE showing expression in E. coli. M = molecular weight markers, T = total protein, and S = supernatant after removal of debris and inclusion bodies. The dense band at 72 kDa representing the unlabeled 30S QconCAT in LB medium is indicated.

Figure 2. Purification and digestion of the 30S QconCAT. (a) SDS-PAGE of the whole inclusion body fraction (SM), material not bound to the His6-tag column (UM), wash fraction (W), and the first and second eluted fractions (1 and 2) all showing the strong band at 72 kDa corresponding to the QconCAT. MALDI mass spectra of tryptic digests of the QconCAT in the presence of (b) RapiGest, (c) 6 M urea/2 M thiourea, and (d) 50 mM NH4HCO3 with no enhancer. ⧫, impurity peaks present in this batch of enhancer and ●, Lys-C-generated peptides.

the intact core QconCAT protein was confirmed by ESI mass spectrometry (data are shown in the Supporting Information, Figure S3). Figure S2 illustrates the extraordinary purity of the core QconCAT in inclusion bodies. The His6-tag column purification was not required; indeed, it contaminated the QconCAT protein.

peptide KLPWR; all other signals derive from Q-peptides intended for quantification. Guanidination22 of lysine side chains to yield homoarginine resulted in substantial improvements in response factors for several of the peptides (Figure S2), although Q4 (the QCAL peptide GVNDNEEGFFSAK) is capable of a second (+42) modification on the N-terminal glycine, as observed by others.23 When the core QconCAT was treated with trypsin following LysC digestion, all of the expected tryptic peptides were detected. Several different digestion protocols were compared, and there was no evidence of missed cleavage in the ribosomal Q-peptides. A small peak resulting from GGVNDNEEGFFSAR (miscleaved QCAL peptide) was observed in some experiments but was not seen once the digestion protocol was optimized. The purity of

30S Ribosome QconCAT

Signature peptides for the 18 small subunit proteins not contained in the core QconCAT were identified experimentally, two peptides for each protein. S6 modification protein (RimK) was also included because it is especially persistent in ribosomal preparations. These signature peptides were inserted into the core QconCAT construct in two groups, Lys-C peptides and 1216



Article

Close inspection of the MALDI spectra (confirmed by MS/ MS measurements) shows that two peptides in the 30S QconCAT are subject to deamidation of asparagine. IFSFTALTVVGDGNGR, [M + H]+ 1659.8 m/z, and LTNGFEVTSYIGGEGHNLQEHSVILIR, [M + H]+ 2989.5 m/z, both contain NG (a sequence known to confer susceptibility in deamidation). Deamidation was not seen in the context of the ribosomal proteins, only in the QconCAT, compromising the use of these sequences for quantification. The importance of redundancy in the design of the QconCAT is illustrated. Both parent proteins (S5 and S12) would now be quantified using their other QconCAT peptide. Three QconCAT proteins were designed with the aim of quantifying 54 ribosomal proteins. After optimizing the digestion of [13C6]arginine/lysine-labeled QconCATs, MALDI-TOF MS analysis of peptides produced complex mass spectra, although most of the signature peptides could be identified by this method (Table 1). When an LTQ Orbitrap or a Q-TOF global mass

tryptic peptides (peptides that could be generated by either enzyme were classed as tryptic), as shown in Figure 1a. In a few cases (for example, SMALRLANELSDAAENK), a Lys-C peptide was chosen that would be expected to be further digested by trypsin. Such a peptide might therefore be used for two or even three independent quantification measurements. The QconCAT protein (approximately 72 kDa) was expressed in E. coli inclusion bodies, as shown in Figure 1b, and purified by affinity chromatography using the His tag. Expression of the QconCAT was optimized with respect to induction time in both LB medium and minimal medium (which is required for the expression of labeled QconCAT). Expression levels were maximized 4 h after induction in LB medium and 5 h after induction in minimal medium, as shown in the Supporting Information (Figure S4). The protein sequence was confirmed by digestion of the gel band corresponding to the QconCAT and MALDI MS (data not shown). A complete list of peptides is shown in Table S3. 50S Ribosomal QconCAT

Table 1. Effects of Enhancers on the Detection of 30S QconCAT Tryptic Peptides by MALDI-TOF MS

Signature peptides for the remaining 30 large subunit proteins were identified experimentally and inserted into the core QconCAT construct in the same way, as shown in the Supporting Information (Figure S5). Because of the high anticipated molecular weight of the recombinant 50S QconCAT, a restriction site was incorporated midway through the construct within a small linker peptide; thus, different peptides for each of the target proteins were separated. This would facilitate subcloning if expression failed. The subsequent amino acid sequences were flanked with a leader N-terminal sequence (MGTK−) and a C-terminal sequence (−LAAALEHHHHHH) ( Supporting Information Figure S5) The 50S QconCAT (approximately 99 kDa) was successfully expressed in the E. coli BL21 strain supplemented with ampicillin. The expression of labeled (Figure S6) and unlabeled 50S QconCAT (data not shown) was carried out for 5 h. SDS-PAGE showed increased 50S QconCAT expression from 2 to 5 h, with the best QconCAT/total protein ratio occurring between 4 and 5 h. 50S QconCAT, labeled and unlabeled, were subjected to in-gel digestion with Lys-C and trypsin, sequentially. The peptide digests were analyzed by MALDI-TOF and nano-LC−MS/MS to confirm the sequence.

digestion enhancer

number of 30S QconCAT peptides detected (out of 36)

6 M urea/2 M thiourea rapigest no enhancer

34 24 29

spectrometer was used for detection with the urea/thiourea digestion method, only two peptides (one each from the 30S and 50S QconCATs) eluded detection. (Table 2). Representative ESI−MS/MS spectra are shown in Figure S11 in the Supporting Information. Table 2. Summary of the Number of Peptides Generated from Lys-C and Trypsin Digests of Labeled 30S and 50S QconCATsa number of peptides observed enzyme

30S QconCAT

50S QconCAT

endopeptidase Lys-C trypsin

16/16 35/36

40/41 33/33

a

Detected using an LTQ Orbitrap MS operating with an inclusion list. The urea/thiourea protocol was used in all digestions. All of the peptides are described in the Supporting Information.

Characterization 30S QconCAT and 50S QconCAT

Like the core QconCAT, the 30S and 50S ribosomal QconCATs were found in inclusion bodies rather than in the soluble fraction of the cell. The inclusion body fractions did not, however, have the outstanding purity of the core QconCAT (see Supporting Information Figures S7 and S8 for SDS-PAGE gels). Additional purification was achieved using a His6-tag column. Both 30S and 50S QconCATs were homogeneous on 1D SDS-PAGE after this step and were used without any other further purification (Figures 2a and S9).

Yields and Concentrations of QconCATS

The three QconCATs were all prepared from 250 mL cultures, and the final purified protein solution had a volume of about 10 mL. The concentrations of QconCAT, as measured by Bradford assay, was 1.8, 0.23, and 0.03 mg mL−1 for core, 30S and 50S QconCATs, respectively, meaning that the yields of these recombinant proteins were 72, 9.2, and 1.2 mg per liter of culture medium. The 30S QconCAT was independently quantified by mass spectrometry using the standard peptide GGVNDNEEGFFSAR, and its concentration was determined to be 0.28 mg mL−1 (11.2 mg per liter of culture medium).

Optimization of QconCAT Digestion

Three common protocols for digestion were applied to the labeled 30S ribosomal QconCAT (Figure 2). Surprisingly, the protocol based on RapiGest was less successful than that based on urea/thiourea for this construct. The urea/thiourea protocol was then applied successfully to digestion of the 50S ribosomal QconCAT (Tables S4 and S5). Guanidination of peptides (Supporting Information, Figure S10 and Table S6) gave rise to improvements in detectability of some peptides, as observed in the case of the ribosomal core QconCAT.

Extent of Labeling

The extent of labeling of several representative peptides for the 30S and 50S QconCATs was measured by MALDI mass spectrometry, choosing peptides with well-separated signals. LysC and tryptic peptides showed average 13C incorporation in the 1217



Article

Table 3. Extent of Incorporation of the 13C Label into the Peptides of the 30S QconCAT Lys-C peptide

percent label

tryptic peptide

percent label

ANLTAQINK* from S20 YQRQLARAIK* from S18 IVPSRITGTRAK* from S18 IRTLQGRVVSDK* from S17 AFNEMQPIVDRQAAK* from S20 AFDHRLIDQATAEIVETAK* from S10

98.0 98.2 97.9 98.3 98.0 98.1

SLEQYFGR* from S9 AYGSTNPINVVR* from S5 FNDAVIR* from S6 LGEFAPTR* from S19 GGGISGQAGAIR* from S9 AGVHFGHQTR* from S2 IGTAITFYGTAAIR* from RimK IAHWVGQGATISDR* from S16 VGFFNPIASEK* from S16 QHVPVFVTDEMVGHK* from S19

98.5 98.3 98.5 98.6 98.5 98.5 98.0 97.9 98.7 98.8

by trypsin (in the presence of 30 pmole of QconCAT) to generate 12 samples (see Experimental Section) and were then subjected to triplicate LC−MS/MS analysis, resulting in 36 LC− MS/MS runs. The absolute amounts of ribosomal proteins under both conditions were determined by comparison with the known amount of 30S QconCAT that was spiked. Automated quantification of the mass spectrometric data was carried out using the QconCAT Pride Wizard. Pride Wizard takes Mascot analysis as its starting point; thus, peptides that are not detected by Mascot cannot be analyzed in this way. Tables 4 and 5 show these data. Additional peptides were analyzed by manual inspection of the MS data. The scatter given by the biological replicates was not significantly greater than that observed in the technical replicates. Each measurement is therefore an average of nine replicates (three technical replicates of each of three biological replicates).

30S QconCAT of 98.1 and 98.4%, respectively (Table 3); the 50S QconCAT was similar (data not shown). It is important that all peptides have similar levels of enrichment. There is a small systematic error in quantification against a QconCAT peptide that is less than 100% enriched, but this is canceled by quantifying the QconCAT by mass spectrometry against a peptide standard. The cancellation works accurately only if all of the QconCAT peptides are enriched to a similar extent. Preparation of Ribosomes from E. coli in the Presence and Absence of Gentamicin

Previous studies of the effect of antibiotics on ribosomal structure revealed that many of the antibiotics that bind to the ribosome and inhibit protein synthesis also bind to partially formed ribosomes, inhibiting ribosomal assembly.7 We have recently shown that treatment of E. coli with gentamicin leads to upregulation of ribosomal proteins,24 but it is not clear whether these proteins are associated with ribosomes or are free in solution. With a QconCAT and a set of digestion protocols in place, we had the means of quantifying ribosomal proteins in real samples. We therefore designed a proof-of-principle experiment to demonstrate that the ribosomal QconCAT can be used for quantification of ribosomal proteins samples. The workflow was adapted from experiments by Mehta and Champney7 on the effect of antiribosomal drugs on bacterial ribosome assembly. Triplicate E. coli cultures were treated with gentamicin when they entered exponential phase (OD600 = 0.2) and harvested after two doublings. The antibiotic concentration was chosen, as described previously,24 to permit two doublings without a significant change in growth rate. Control cultures were grown in the same way without the addition of gentamicin. Ribosomes were isolated from 200 mL of cell culture at an OD600 of 0.8 (control) or 0.6 (gentamicin-treated) cells, and the resulting pellet was dissolved in 2 mL of buffer. The concentration of ribosomes was estimated by withdrawing 10 μL, diluting to 1 mL, and measuring the OD260. The values obtained were 0.445 for the control and 0.207 for the gentamicintreated cultures, indicating that 1 mL of the original culture contained 10.7 and 5.0 pmole ribosomes, respectively.

Accuracy and Precision

Each of the 21 proteins of the 30S subunit gave quantitative data for at least one signature peptide, and quantification showed good agreement between the technical and biological triplicates (see standard errors in Table 4, which are typically 3%). (There was some signal overlap for some of the peptides, preventing their quantification, and this could, if necessary, be overcome using MRM measurements.) Where more than one peptide can be used for quantification, there is very good agreement between measurements. Where the two measurements are derived from the use of the same enzyme (trypsin for S9, S11, S15, S16, S21, and L4 and Lys-C for S7 and L13), the measurements are completely consistent with one another. Measurements for S8, S10, and S13 (one or more from each enzyme) are also consistent. Only S20 shows a discrepancy, of about 15%, between the Lys-C and trypsin measurements. Stoichiometries of Ribosomal Proteins in Control and Gentamicin-Treated Samples

Proteins L2, L4, and L13 were chosen as central ribosomal proteins for the construction of the core QconCAT and are on the large ribosomal subunit, remote from the gentamicin binding site. The stoichiometries of the ribosomal proteins in the impure ribosome samples were now determined by reference to the average amounts of these three proteins. Figure 3 shows that in the control samples there was very little deviation from 1:1:1 in the 30S ribosomal proteins with the excepts that ribosomal protein S1 is strikingly underrepresented and S2 is abundant compared with the other proteins in the samples. This is quite consistent with the literature.22 Although purified single ribosomes appear to contain one copy of S1 and one copy of S2, the overall copy number of S1 is low and that of S2 is high.

Quantification of 30S Ribosomal Proteins in Control and Gentamicin-Treated E. coli Ribosomes

E.coli 70S ribosomes (52.2 μg, 22.6 pmole, as estimated from OD260 measurements) were first isolated from the whole lysate of both control and gentamicin-treated samples in biological triplicate and then spiked with 60 or 30 pmole of 30S labeled QconCAT. Three control and three gentamicin-treated ribosome samples were digested with Lys-C (in the presence of 60 pmole of QconCAT) and separately with Lys-C followed 1218



Article

Table 4. Quantification of Peptides from Control and Gentamicin-Treated E. coli Ribosomesa protein

peptide

S1

AFLPGSLVDVR*PVR* GATVELADGVEGYLR* AGVHFGHQTR* LENSLGGIK* ADIDYNTSEAHTTYGVIGVK* LGIVKPWNSTWFANTK* LDNVVYR* SDLSADINEHLIVELYSK* AYGSTNPINVVR* IFSFTALTVVGDGNGR* FNDAVIR* YTAAITGAEGK* LANELSDAAENK* SMALR*LANELSDAAENK* FGSELLAK* AVVESIQR* VEGDTK*PELELTLK* GGGISGQAGAIR* SLEQYFGR* LIDQATAEIVETAK* LVDIVEPTEK* ALNAAGFR* QGNALGWATAGGSGFR* VYTTTPK* LTNGFEVTSYIGGEGHNLQEHSVILIR* IAGINIPDHK* ISELSEGQIDTLR*DEVAK* AIISDVNASDEDR* AIISDVNASDEDR*WNAVLK* IVSEFGR* YTQLIER* VGFFNPIASEK* IAHWVGQGATISDR* SIVVAIER* IR*TLQGR*VVSDK* YQR*QLAR*AIK* IVPSR*ITGTR*AK* LGEFAPTR* QHVPVFVTDEMVGHK* AFNEMQPIVDR* AFNEMQPIVDR*QAAK* ANLTAQINK* AGVLAEVR* EFYEK*PTTER* SAGTYVQIVAR* LEYDPNR* DAQSALTVSETTFGR* DFNEALVHQVVVAYAAGAR* TFTAK*PETVK* R*DWYVVDATGK*

S2 S3 S4 S5 S6 S7

S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20

S21 L2 L4 L13

m/z

z

score

control L/H ratio ± SE

gentamicin-treated L/H ratio ± SE

769.46 778.40

2 2 2 2 2

24.7 45.5

0.59 ± 0.042 OL OL 2.65 ± 0.06 1.68 ± 0.023 ND 1.02 ± 0.006 OL 1.28 ± 0.019 1.29 ± 0.021 1.19 ± 0.054 OL 1.37 ± 0.083 1.31 ± 0.03 1.32 ± 0.05 1.26 ± 0.031 1.14 ± 0.07 1.17 ± 0.055 1.20 ± 0.097 1.26 ± 0.063 1.27 ± 0.041 1.40 ± 0.057 1.45 ± 0.155 1.19 ± 0.046 ND 1.29 ± 0.106 1.31 ± 0.04 MC 1.44 ± 0.04 1.32 ± 0.018 1.30 ± 0.029 1.66 ± 0.038 1.65 ± 0.072 1.22 ± 0.004 1.30 ± 0.11 1.88 ± 0.01 1.71 ± 0.1 1.30 ± 0.090 OL 2.11 ± 0.007 1.88 ± 0.08 1.61 ± 0.24 1.04 ± 0.11 1.15 ± 0.16 1.25 ± 0.036 OL 1.57 ± 0.070 OL 1.33 ± 0.16 1.32 ± 0.09

1.01 ± 0.065 OL OL 4.47 ± 0.09 2.38 ± 0.015 ND 1.79 ± 0.084 OL 2.23 ± 0.060 OL 1.81 ± 0.027 OL 2.28 ± 0.121 2.44 ± 0.03 2.22 ± 0.04 OL 1.10 ± 0.01 2.25 ± 0.044 1.81 ± 0.035 2.20 ± 0.009 2.08 ± 0.141 2.30 ± 0.175 2.39 ± 0.093 1.35 ± 0.108 ND 2.38 ± 0.102 2.15 ± 0.15 MC 2.24 ± 0.03 2.45 ± 0.054 OL 2.31 ± 0.13 2.32 ± 0.110 2.11 ± 0.004 1.93 ± 0.39 1.67 ± 0.22 1.68 ± 0.18 1.93 ± 0.176 OL 2.94 ± 0.079 2.63 ± 0.05 2.40 ± 0.02 0.88 ± 0.22 1.13 ± 0.07 2.03 ± 0.09 OL 2.42 ± 0.096 2.32 ± 0.20 2.08 ± 0.16 2.09 ± 0.07

468.77 1080.5 442.74 684.83 648.85 830.43 420.74 544.29 640.82 922.98 435.75 454.26 792.45 525.29 503.26 754.42 574.83 413.23 778.39 408.20 ND 542.31 1008.03 MC 1064.5 407.23 464.75 607.83 758.90 446.78 695.44 422.25 433.76 448.74 861.93 663.34 865.45 489.78 410.75 656.3 585.83 456.73 794.90 679.36 567.33 661.35

45.2 99.4 ND

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 3 2 2

50.2 50.1 41.4 77.8 80.8 49.4 19.9 78.9 93.3 28.6 21.1 52.3 56.1 104.6 30.2 ND 53.5 75.1 MC 101.2 39.1 57.1 64.1 73.4 54.91 32.6 25.7 15.2 26 34.3 61.1 19.2 15.9 18.5 45.3 66.2 42.2 93.7 24.6 49.3 68.9

a

Each data point represents an average of nine replicates, three technical replicates of each of three biological replicates. Because the biological replicates did not add to the scatter of the data, all of the replicates were combined and treated equally. Peptides generated using Lys-C are in bold, and data were normalized to 30 pmole of QconCAT by doubling the L/H ratio; those generated with trypsin are in normal type face. Data shown in italic were obtained manually, and others were obtained using Pride Wizard. A Mascot score of above 20 represents above 95% confidence in the identification. OL = overlapped, MC = miscleaved, and ND = not detected.

concentration to the control samples. The exceptions are S21, S18, S12, and, most especially, S8, of which all are underrepresented. The mean relative concentration of the ribosomal S proteins compared with L2, L4, and L13 is now 0.96 ± 0.34

When compared with the reference L proteins, the mean relative concentration of the ribosomal S proteins is 1 ± 0.29 (SD); when S1 and S2 are excluded, the standard deviation falls to 0.16. For the samples treated with gentamicin, Figure 3 shows that most of the ribosomal proteins measured are present at a similar 1219



Article

pmole (gentamicin-treated) of ribosomes. The expected number of E. coli cells in 1 mL of culture at an OD600 of 1 in Luria broth is 7.8 × 108 (6.24 × 108 for controls at OD600 of 0.8, 4.68 × 108 for gentamicin-treated cultures at OD600 of 0.6).26 On the basis of these values, the estimated average copy number for L2, L4, and L13 is 18 800 for control and 18 300 for gentamicin-treated samples, and overall errors are estimated at 20%. Table 5 shows the calculated copy number per cell of ribosome-associated protein for each protein measured.

Table 5. Estimated Copy Numbers Per Cell of RibosomeAssociated E. coli Ribosomal Proteins in the Presence and Absence of Gentamicin copy number per cell of ribosome-associated protein ribosomal protein

control sample

gentamicin-treated

L2 L4 L13 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

17 100 21 500 18 100 8100 36 200 23 000 13 900 17 500 16 200 18 300 16 400 16 200 17 100 19 600 16 200 17 700 19 600 17 900 22 600 17 100 24 500 17 700 24 700 15 100

17 200 20 100 17 600 8600 37 900 20 100 15 200 18 800 15 400 19 400 9300 15 400 17 900 19 900 11 500 19 200 19 000 20 700 19 600 17 200 14 100 16 300 22 000 8600

■

DISCUSSION Ribosomes are ribonucleoprotein complexes of more than 2.3 MDa and are responsible for the translation of genetic information into proteins. Protein synthesis can consume up to 40% of the energy turnover of a bacterial cell, making the ribosome a primary subject of research for well over 100 groups worldwide. Numerous elegant studies of ribosome structure, function, and assembly have, however, generally employed purified ribosomes and while much is known about ideal ribosomes and their biogenetic precursors, much is less is known about the total ribosomal material in the cell. The work of Williamson and co-workers27 is notable for its successful use of mass spectrometry with isotopically labeled ribosomal components in the investigation of ribosome biogenesis. Here, we extend that approach with the generation of isotopically labeled standards (QconCATs) that can be used to determine absolute concentrations of ribosomal proteins and, more importantly, their stoichiometries within whole cells and cellular fractions. QconCAT methodology dates from 200511 and is especially powerful for quantification of tens or hundreds of proteins, and where many experiments are planned. For single experiments with small numbers of proteins, an approach based on standard synthetic peptides is much simpler,28 and where an entire cellular proteome is under study, label-free methods are currently the most reasonable option. The translation machinery presents particular difficulties for label-free quantification. The ribosomal proteins are small and basic, but they interact with proteins such as EFTu and EFG, which are much larger and more acidic; labelfree methods for determining stoichiometries all depend, to an extent, on the proteins being compared having some similar properties. Methods that introduce a labeled standard peptide, including QconCAT, do not suffer from this limitation. We therefore designed a core QconCAT that can be elaborated in a relatively straightforward way to allow many different proteins to be quantified relative to the central ribosomal proteins. We have successfully elaborated it to give the 30S and 50S QconCATs. Because the ribosomal proteins are small and basic, a two-enzyme strategy was required and developed to permit quantification of all of these proteins simultaneously. This strategy significantly reduces missed cleavages, which can complicate QconCAT design. Endoproteinase Lys-C cleaves at lysine but not at arginine, and this directs cleavage in richly basic regions of a protein. All three QconCATs expressed adequately in E. coli inclusion bodies, but the yields are starkly different. The small core QconCAT is expressed with a yield of 8 and 60 times the yields of the 30S and 50S QconCATs (more if the molar yield is considered). There is substantial anecdotal evidence that small QconCATs express far better than larger ones, and these numerical results are in agreement. Incorporation of label from 13 C6-argine and -lysine is uniformly good across the three QconCATs and varies little between the peptides under study.

Figure 3. Stoichiometries of ribosomal proteins using 30S QconCAT. The amount of each protein is normalized relative to the mean of L2, L4, and L13. Untreated samples are indicated in blue, and gentamicintreated, in red.

(SD); when S1 and S2 are excluded, the standard deviation falls to 0.21. Absolute Quantification of 30S Ribosomal Proteins

Table 4 shows that in the control sample the average L/H ratio for L2, L4, and L13, on the basis fo 30 pmole of QconCAT, is 1.38 ± 0.17. Thus, the absolute amounts of these proteins in the analyzed sample is 41.4 ± 5 pmole. For the gentamicin-treated sample, the average L/H ratio is 2.16 ± 0.18, corresponding to 64.8 ± 5.4 pmole. The analyzed sample originated from 2.12 (control) and 4.55 mL (gentamicin-treated) of culture, so 1 mL of cell culture contained 19.5 ± 2.40 (control) and 14.2 ± 1.18 1220



Article

designed to quantify human cytochrome P450 enzymes.30 The hydrophobic 30S QconCAT forces the whole construct to express in inclusion bodies, protecting the fusion partner from proteolytic degradation. It seems likely, however, that the smaller core QconCAT will ultimately be preferable for this purpose. In conclusion, flexible tools for the quantification of the components of the E. coli protein synthesis machinery were developed.

This is clearly important for quantification, and the protocols resulting in these levels (>98%) represent the wisdom and expertise of many researchers in different groups, informally combined. The concept of a flexible core QconCAT is, we believe, novel. It lends itself especially well to the translational machinery, where quantification of hundreds of proteins may be desirable. The core QconCAT was chosen to quantify proteins that can stand proxy for ribosomes themselves. L2, L4, L13, S4, S7, and S8 are central proteins that are assembled early. The core QconCAT expresses exceptionally well in inclusion bodies and represents a good starting point for the quantification of proteins involved in translation. There is excellent agreement between technical replicates and biological replicates of the same peptide, with standard errors normally below 3%. Even where two different peptides were used to quantify the same protein, the measurements were consistent except for a single protein, S20, where the difference was about 15%. The control samples vindicate our choice of core proteins. The measured levels of L2, L13, S7, and S8 differed by less than 10%. L4 and S4 were outliers, with L4 registering 20% above and S4 (for which manual measurements were necessary) 22% below the mean amounts of the other four proteins. The concentration of S1 was low and that of S2 was high relative to the other proteins of the 30S subunit, and this is consistent with the literature.25 Given the very high reproducibility of our data, it is reasonable to conclude that the concentrations of S18 and S20 are also elevated but by smaller amounts. The selectivity and sensitivity of the QconCAT method allows ribosomal proteins to be quantified relative to one another so that stoichiometries may be determined. The approximate number of ribosomes in an E. coli cell is reported to be 20 000.29 The value of 18 800 for ribosomeassociated L-proteins is completely consistent. This consistency, together with the internal consistency of the data, suggests that the ribosomal QconCATs might additionally be used for quantification of ribosomes. OD260 measurements15 are very rapid and straightforward but are not very selective. Quantification using a QconCAT takes hours rather than seconds, but it is specific, internally validated (several proteins are quantified in the same experiment), and not dependent on the purity of the ribosomes. We have previously used label-free methods to investigate the effects of the protein synthesis inhibitor, gentamicin, on the whole E. coli proteome.21 Several ribosomal proteins were upregulated in the presence of the drug. The present work shows, however, that the gentamicin-treated ribosomal fraction contains very similar amounts of most of the ribosomal proteins compared to the control. The amount of gentamicin used was intended to cause minimal perturbation to the growth of the E. coli cultures in early log phase, so this is perhaps not a surprising result. It suggests that the upregulated proteins (S1 and S2 are notable)21 accumulate in the cytosol rather than in the ribosomal fraction. Gentamicin also appears to cause under-representation of S8, S12, S18, and S21 in the ribosome fraction, and this is consistent with the work of Mehta and Champney,7 who have shown that aminoglycosides and other antiribosomal antibiotics disrupt the assembly of bacterial ribosomes. A third use for the ribosomal QconCATs is as fusion partners. These proteins accumulate in relatively pure form in inclusion bodies. The 30S ribosomal QconCAT has already found utility as a hydrophobic fusion partner for a recalcitrant QconCAT

■

ASSOCIATED CONTENT

* Supporting Information S

Numbers of peptides resulting from theoretical digests of ribosomal proteins using Protein Prospector and various proteolytic enzymes; DNA sequence of the synthetic gene (core QconCAT); core QconCAT sequence showing Lys-C peptides, tryptic peptides, and sacrificial peptides; SDS-PAGE showing expression in E. coli; MALDI mass spectrum of Lys-C digest of the core QconCAT before and after guanidination; MALDI mass spectrum of the sequential Lys-C/tryptic digest of the core QconCAT; core ribosomal QconCAT showing origins of the proteolytic peptides; expression of unlabeled 30S QconCAT in LB medium, unlabeled QconCAT in minimal medium, and labeled QconCAT in minimal medium; signature peptides in the 30S ribosomal subunit QconCAT; 50S ribosomal QconCAT sequence showing Lys-C peptides, tryptic peptides, and sacrificial peptides containing restriction enzymes; expression of labeled 50S QconCAT in minimal medium; expression of the 30S and 50S QconCAT in the total and soluble fractions of transformed E. coli cells; His-tag purification of the 50S-labeled QconCAT; LC−MS/MS Mascot results from a single LC−MS/MS analysis of a Lys-C digest and a tryptic digest of the 50S ribosomal QconCAT using a LTQ-Orbitrap mass spectrometer XL; MALDI-TOF mass spectra of 50S QconCAT labeled Lys-C digest; 50S ribosomal QconCAT peptides; and Q-TOF CID MS/MS spectra of the doubly protonated peptides ion, TYAAITGAEGK (S6), at m/z 544.24. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +44 (0) 161 275 2369. Fax: +44 (0) 161 275 2396. Email: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Professor Kathryn Lilley and Dr. Ruth Ledder for their constructive review of the draft manuscript. We are particularly grateful to Dr. Neil Swainston for help with the use of the Pride wizard.

■

ABBREVIATIONS USED E. coli, Escherichia coli; CID, collision-induced dissociation; LysC, endopeptidase Lys-C; LB, Luria broth; Th, thomson; mgf, Mascot generic file

■

REFERENCES

(1) Wilson, D. N.; Nierhaus, K. H. The Weird and Wonderful World of Bacterial Ribosome Regulation. Crit. Rev. Biochem. Mol. Biol 2007, 42, 187−219.

1221



Article

(2) Poehlsgaard, J.; Douthwaite, S. The Bacterial Ribosome as a Target for Antibiotics. Nat. Rev. Microbiol 2005, 3, 870−881. (3) Wimberley, B. T.; Brodersen, D. E.; Clemons, W. M., Jr.; MorganWarren, R. J.; Carter, A. P.; Vonrhein, C.; Hartsch, T.; Ramakrishnan, V. Structure of the 30S Ribosomal Subunit. Nature 2000, 407, 327−339. (4) Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution. Science 2000, 289, 905−920. (5) Schleunzen, F.; Tocilj, A.; Zarivach, R.; Harms, J.; Gluehmann, M.; Janell, D.; Bashan, A.; Bartels, H.; Agmon, I.; Franceschi, F.; Yonath, A. Structure of Functionally Activated Small Ribosomal Subunit at 3.3 Angstroms Resolution. Cell 2000, 102, 615−623. (6) Usary, J.; Champney, W. S. Erythromycin Inhibition of 50S Ribosomal Subunit Formation in Escherichia coli Cells. Mol. Microbiol. 2001, 40, 951−962. (7) Mehta, R.; Champney, W. S. 30S Ribosomal Subunit Assembly Is a Target for Inhibition by Aminoglycosides in Escherichia coli. Antimicrob. Agents Chemother. 2002, 46, 1546−1549. (8) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Mol. Cell. Proteomics 2002, 1, 376−386. (9) Multiplex Protein Quantitation using iTRAQ® Reagents−8plex. http://tools.lifetechnologies.com/content/sfs/manuals/cms_049786. pdf (accessed October 13, 2009). (10) Kirkpatrick, D. S.; Gerber, S. A.; Gygi, S. P. The Absolute Quantification Strategy: A General Procedure for the Quantification of Proteins and Post-Translational Modifications. Methods 2005, 35, 265− 273. (11) Beynon, R. J.; Doherty, R. J.; Pratt, J. M.; Gaskell, S. J. Multiplexed Absolute Quantification in Proteomics Using Artificial QCAT Proteins of Concatenated Signature Peptides. Nat. Methods 2005, 2, 587−589. (12) Protein Prospector; University of California at San Francisco: San Francisco, CA; http://prospector.ucsf.edu/prospector/mshome.htm. (13) Wierenga, S. K.; Zocher, M. J.; Mirus, M. M.; Conrads, T. P.; Goshe, M. B.; Veenstra, T. D. A Method to Evaluate Tryptic Digestion Efficiency for High-Throughput Proteome Analyses. Rapid Commun. Mass Spectrom. 2002, 16, 1404−1408. (14) Mann, M. In solution digest for mass spectrometry (MS) analysis; RUBICON Protocols Database. (15) Nierhaus, K. H. Reconstitution of Ribosomes. In Ribosomes and Protein Synthesis: A Practical Approach; Spedding, G., Ed.; Oxford University Press: New York, 1990; Chapter 8. (16) Carroll, K. M.; Simpson, D. M.; Eyers, C. E.; Knight, C. G.; Brownridge, P.; Dunn, W. B.; Winder, C. L.; Lanthaler, K.; Pir, P.; Malys, N.; Kell, D.b.; Oliver, S. G.; Gaskell, S. J.; Beynon, R. J. Absolute Quantification of the Glycolytic Pathway in Yeast: Deployment of a Complete QconCAT Approach. Mol. Cell. Proteomics 2011, 10, M111.007633-1−M111.007633-15. (17) Swainston, N.; Jameson, D.; Carroll, K. A QconCAT Informatics Pipeline for the Analysis, Visualization and Sharing of Absolute Quantitative Proteomics Data. Proteomics 2011, 11, 329−333. (18) Savitzky, A.; Golay, M. J. E. Smoothing and Differentiation of Data by Simplified Least Squares Procedures. Anal. Chem. 1964, 36, 1627−1639. (19) Al-Majdoub, Z. M. Quantification of Ribosomal Proteins by Mass Spectrometry: An Approach to Drug Target Identification. Ph.D. Thesis, The University of Manchester, May 2013, pp 93−104. (20) Washburn, M. P.; Wolters, D.; Yates, J. R., III Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology. Nat. Biotechnol. 2001, 19, 242−247. (21) Eyers, C. E.; Simpson, D. M.; Wong, S. C.; Beynon, R. J.; Gaskell, S. J. QCAL − a Novel Standard for Assessing Instrument Conditions for Proteome Analysis. J. Am. Soc. Mass Spectrom. 2008, 19, 1275−1280. (22) Warwood, S.; Mohammed, S.; Cristea, I. M.; Evans, C.; Whetton, A. D.; Gaskell, S. J. Guanidination Chemistry for Qualitative and Quantitative Proteomics. Rapid Commun. Mass Spectrom. 2006, 20, 3245−3256.

(23) Beardsley, R. L.; Reilly, J. P. Optimization of Guanidination Procedure for MALDI Mass Mapping. Anal. Chem. 2002, 74, 1884− 1890. (24) Al-Majdoub, Z. M.; Owenseni, A.; Gaskell, S. J.; Barber, J. Effects of Gentamicin on the Proteomes of Aerobic and Oxygen-Limited Escherichia coli. J. Med. Chem. 2013, 56, 2904−2910. (25) Van Knippenberg, P. H.; Hooykaas, P. J.; Van Duin, J. The Stoichiometry of E. coli Ribosomal Protein S1 on in Vivo and in Vitro Polyribosomes. FEBS Lett. 1974, 41, 323−326. (26) Volkmer, B.; Heinemann, M. Condition-dependent cell volume and concentration of Escherichia coli to facilitate data conversion for systems biology modelling. PLoS ONE 2011, 6, e23126-1−e23126-6. (27) Chen, S. S.; Williamson, J. R. Characterization of the Ribosome Biogenesis Landscape in E. coli using Quantitative Mass Spectrometry. J. Mol. Biol. 2013, 425, 767−779. (28) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Absolute Quantification of Proteins and Phosphoproteins from Cell Lysates by Tandem MS. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940− 6945. (29) British Society for Cell Biology Ribosome Page. http://www.bscb. org/?url=softcell/ribo (accessed December 6, 2013). (30) Russell, M. R.; Achour, B.; McKenzie, E. A.; Lopez, R.; Harwood, M. D.; Rostami-Hodjegan, A.; Barber, J. Alternative fusion protein strategies to express recalcitrant QconCAT proteins for quantitative proteomics of human drug metabolizing enzymes and transporters. J. Proteome Res. 2013, 12, 5934−5942.

1222


Quantification of the Proteins of the Bacterial Ribosome Using

Recommend Documents