Physiological Adaptation of the Bacterium Lactococcus lactis in Response to the Production of Human CFTR
2010; Elsevier BV; Volume: 10; Issue: 7 Linguagem: Inglês
10.1074/mcp.m000052-mcp201
ISSN1535-9484
AutoresAnton Steen, Elena Wiederhold, Tejas Gandhi, Rainer Breitling, Dirk Jan Slotboom,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoBiochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that different strategies are needed to overcome low expression yields and toxicity. Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that different strategies are needed to overcome low expression yields and toxicity. The human cystic fibrosis transmembrane conductance regulator CFTR 1The abbreviations used are:CFTRcystic fibrosis transmembrane conductance regulatorDDMn-dodecyl-β-D-maltosideiTRAQisobaric tag for relative and absolute quantificationMALDImatrix assisted laser desorption/ionisationMBPmaltose binding proteinMMTSmethyl methanethiosulfonatePMSFphenyl methanesulphonyl fluorideTEABtriethylammonium bicarbonate bufferTFAtrifluoroacetic acidTOFtime-of-flight. is an atypical member of the superfamily of ATP binding cassette (ABC) transporters, because it is a channel (for chloride ions) rather than a transporter. Mutations in CFTR cause cystic fibrosis (1Rommens J.M. Iannuzzi M.C. Kerem B. Drumm M.L. Melmer G. Dean M. Rozmahel R. Cole J.L. Kennedy D. Hidaka N. Zsiga M. Buchwald M. Riordan J.R. Lap C.T. Collins F.S. Identification of the cystic fibrosis gene: chromosome walking and jumping.Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2555) Google Scholar, 2Kerem B. Rommens J.M. 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In an attempt to find suitable hosts for recombinant production of CFTR the cystic fibrosis foundation has funded a project to express CFTR in the bacterium Lactococcus lactis. cystic fibrosis transmembrane conductance regulator n-dodecyl-β-D-maltoside isobaric tag for relative and absolute quantification matrix assisted laser desorption/ionisation maltose binding protein methyl methanethiosulfonate phenyl methanesulphonyl fluoride triethylammonium bicarbonate buffer trifluoroacetic acid time-of-flight. L. lactis is a Gram-positive bacterium for which expression plasmids and inducible promoters are available (12de Ruyter P.G. Kuipers O.P. de Vos W.M. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin.Appl. Environ. Microbiol. 1996; 62: 3662-3667Crossref PubMed Google Scholar). Several cases have been reported in which functional overexpression of membrane proteins could be achieved in L. lactis, but not in E. coli (e.g. the human KDEL receptor, Na+/tyrosine transporter (Tyt1) of Fusobacterium nucleatum and several membrane proteins from Arabidopsis) (5Kunji E.R. Slotboom D.J. Poolman B. Lactococcus lactis as host for overproduction of functional membrane proteins.Biochim. Biophys. Acta. 2003; 1610: 97-108Crossref PubMed Scopus (163) Google Scholar, 13Frelet-Barrand, A., Boutigny, S., Moyet, L., Deniaud, A., Seigneurin-Berny, D., Salvi, D., Bernaudat, F., Richaud, P., Pebay-Peyroula, E., Joyard, J., Rolland, N., Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins. PLoS One 5, e8746Google Scholar, 14Kunji E.R. Chan K.W. Slotboom D.J. Floyd S. O'Connor R. Monné M. Eukaryotic membrane protein overproduction in Lactococcus lactis.Curr. Opin. 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Biotechnol. 2005; 16: 546-551Crossref PubMed Scopus (56) Google Scholar). Among the potential advantages of L. lactis are its growth rate of ∼1 doubling per hour, which is much slower than E. coli and could be beneficial for expression of proteins that do not fold easily. Also the presence of a different repertoire of chaperones, e.g. two copies of the integral membrane chaperone YidC (18Luirink J. Samuelsson T. de Gier J.W. YidC/Oxa1p/Alb3: evolutionarily conserved mediators of membrane protein assembly.FEBS Lett. 2001; 501: 1-5Crossref PubMed Scopus (123) Google Scholar, 19Zweers J.C. Barák I. Becher D. Driessen A.J. Hecker M. Kontinen V.P. Saller M.J. Vavrová L. van Dijl J.M. Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes.Microb. Cell Fact. 2008; 7: 10Crossref PubMed Scopus (99) Google Scholar, 20Funes S. Hasona A. Bauerschmitt H. Grubbauer C. Kauff F. Collins R. Crowley P.J. Palmer S.R. Brady L.J. Herrmann J.M. Independent gene duplications of the YidC/Oxa/Alb3 family enabled a specialized cotranslational function.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 6656-6661Crossref PubMed Scopus (61) Google Scholar), could facilitate insertion and assembly of heterologous membrane proteins. Other factors such as different membrane lipids and cytosolic environment could play a role as well. Here we have used L. lactis for the expression of the human cystic fibrosis transmembrane conductance regulator CFTR. We were able to express full length (1480 amino acids long) CFTR in the bacterial host, but the expression levels were too low for to pursue structural studies, and expression was toxic to the cells. To understand this toxicity and to identify potential remedies to improve expression levels, we investigated the physiological responses that were elicited in L. lactis upon CFTR expression by performing a global quantitative proteomics study. Human cftr cDNA (gift from Christine Bear, Toronto) (accession nr M28668) was cloned into the E. coli vectors pREnLIC, and pREcLIC (supplemental Table 1), which introduce the sequences coding for N- and C-terminal His10-tags, respectively, by Ligation Independent Cloning (LIC) as described by Geertsma et al. (21Geertsma E.R. Poolman B. High-throughput cloning and expression in recalcitrant bacteria.Nat. Methods. 2007; 4: 705-707Crossref PubMed Scopus (85) Google Scholar), yielding plasmids pREnCFTR and pREcCFTR. Plasmid pREncCFTR, which contains the cftr coding region fused to sequences coding for both an N- and C-terminal His10-tag, was constructed by exchanging the NcoI-XhoI fragment of pREnCFTR with the NcoI-XhoI fragment of pREcCFTR. pRE_MBP-CFTR was constructed by amplifying cftr by PCR and subsequent cloning in the NcoI and SpeI sites of pRE_MBP (supplemental Table 1). The pRE vectors were converted into pNZ8048-related vectors for L. lactis by Vector Backbone Exchange as described by Geertsma et al. (21Geertsma E.R. Poolman B. High-throughput cloning and expression in recalcitrant bacteria.Nat. Methods. 2007; 4: 705-707Crossref PubMed Scopus (85) Google Scholar). L. lactis NZ9000 transformed with pNZ8048-derived plasmids was cultivated in M17 medium (Oxoid, Basingstoke, UK) containing 1% glucose, and 5 μg/ml chloramphenicol. To test for expression of CFTR L. lactis was grown in 10-ml cultures (inoculated with O/N cultures that were diluted 1:50) to an OD600 of 0.5 at 30 °C, after which Nisin A (1:5000 dilution of the culture supernatant of the nisin producing strain L. lactis NZ9700 (5Kunji E.R. Slotboom D.J. Poolman B. Lactococcus lactis as host for overproduction of functional membrane proteins.Biochim. Biophys. Acta. 2003; 1610: 97-108Crossref PubMed Scopus (163) Google Scholar)) was added and the cells were incubated for another 2 h. A volume of culture containing the equivalent amount of cells as 1 ml of OD600 of 5 was spun down (20,000 × g, 2 min, room temperature) and the pellet was resuspended in 400 μl of 50 mm potassium phosphate buffer (KPi) pH 7.5, 10% glycerol. Phenyl methanesulphonyl fluoride (1 mm) was added and the cells were disrupted in a Fastprep machine (Bio101, Vista, CA) by vigorous shaking in the presence of glass beads (two times at force 6.0 for 30 s, with 10 min incubation on ice in between the two runs). The crude cell extracts were supplemented with EDTA (15 mm final concentration) and centrifuged for 15 min at 20,000 × g at 4 °C. The supernatant was subsequently centrifuged at 300,000 × g (30 min, 4 °C) to obtain the membranes. SDS-sample buffer was added and samples were incubated at 37 °C for 5 min before loading on SDS-PAGE. His-tag specific antibodies (GE healthcare) and CFTR C terminus specific antibodies (clone 24–1, R&D systems) were used for western hybridizations. L. lactis NZ9000 pNZ_MBP-CFTR was grown in a fermenter (Applikon) in 2 L M17 supplemented with glucose (1%) and chloramphenicol (5 μg/ml) as described later. The cells were harvested 2 h after induction by centrifugation (6800 × g, 15 min, 4 °C) and membranes were prepared as described later. The membranes were stored at −80 °C in 50 mm KPi pH7.5, 10% glycerol at a concentration of 10 mg/ml protein. Membranes containing 10 mg protein were resuspended in 10 ml of 50 mm Tris-HCl pH 8.0, 300 mm NaCl, 20% glycerol, 10 mm Imidazole. n-Dodecyl-β-d-maltoside (DDM) was added (1% final concentration) and the proteins were solubilized on ice for 1 h. Solubilized membranes were centrifuged at 100,000 × g for 30 min at 4 °C. The supernatant was incubated with Ni-Sepharose resin (GE Healthcare) for 1 h (400 μl slurry, which had been pre-equilibrated with solubilization buffer), with gentle rotation. The resin was washed with 10 ml of the same buffer containing 0.05% DDM and 50 mm imidazole and finally proteins were eluted with buffer containing 500 mm imidazole and 0.05% DDM (100, 200, 200 μl fractions). Bands were excised from a Coomassie Blue-stained SDS-PAGE and cut into ∼1 mm3 pieces. Gel slices were incubated 3–4 times for 15 min in 150 μl of destaining solution (50% acetonitrile, 50 mm ammonium bicarbonate). The gel slices were dehydrated in 150 μl of 100% acitonitrile for 10 min, the supernatant was discarded and the gel slices were dried by evaporation. The reduction of cysteine residues was performed by incubating the gel slices in 30 μl of 10 mm dithiotreitol in 50 mm ammonium bicarbonate for 45 min at 55 °C. The supernatant was removed, 30 μl of 55 mm iodoacetamide in 50 mm ammonium bicarbonate was added to each gel slice and incubated for 30 min at RT. The gel slices were dehydrated as above. To each dried gel slice, 7–10 μl of 10 ng/μl trypsin gold (Cat.: V5280, Promega, Madison, WI) in 40 mm ammonium bicarbonate/10% acetonitrile were added and allowed to re-swell for ∼20 min at 37 °C. The gel slices were overlaid with 20 μl of 40 mm ammonium bicarbonate, 10% acetonitrile and incubated overnight at 37 °C. The peptides were extracted by adding 50 μl of 2% trifluoroacetic acid (TFA) to each gel slice without removing the overlay. The extraction was repeated twice with 33% acetonitrile/1.3% TFA and 63% acetonitrile/0.7% TFA. The extracted peptides were combined and the peptide mixture was dried. The peptide mixture was resuspended in 10 μl of 0.1% TFA and subjected to tandem MS (MS/MS) analysis directly by the mixing 1:2 with 20 mg/ml α-cyano-4-hydroxycinnamic acid matrix solution (LaserBio Labs, Sophia-Antipolis, France) onto a matrix assisted laser desorption ionization (MALDI) target. L. lactis NZ9000 pNZ8048 and L. lactis NZ9000 pNZncCFTR were grown in 3 L fermenters (Applikon) in M17 medium supplemented with glucose (1%) and chloramphenicol (5 μg/ml). The temperature was set at 30 °C and the pH was maintained at 6.5 during growth by addition of KOH. At an OD600 of 0.5 900 ml of the culture was removed and to the remaining culture Nisin A was added (1:5000 dilution of the supernatant of a culture of L. lactis NZ9700). After 1 h and 4 h of induction 900 ml of the culture was collected. Cell were spun down (6800 × g for 15 min, 4 °C), and pellets were washed once with 10 mm KPi pH 7.5. The washed cell pellets were frozen in liquid nitrogen and stored at −80 °C. The cell pellets were resuspended in 10 mm KPi pH 7.5 at an OD600 of 50. To 6 ml of the suspension MgCl2 was added (1 mm final concentration) and the cells were disrupted at 39 kPsi with a Constant Systems cell disrupter. The cells were passed through the disrupter cell twice. EDTA was added (15 mm) to the suspensions and they were incubated on ice for 15 min. To remove nonbroken cells the crude cell lysates were centrifuged for 15 min at 12,000 × g at 4 °C. The supernatant was carefully recovered and subsequently centrifuged at 267,000 × g for 15 min at 4 °C. The supernatant, containing the soluble protein fraction was carefully pipetted off and stored at −80 °C. Residual supernatant was completely removed from the membranes pellet. The membranes were washed once with 1 ml 10 mm KPi containing 10% glycerol. The pellets were finally resuspended in 500 μl 10 mm KPi, 10% glycerol and stored at −80 °C. Protein concentrations were determined with the BCA kit (Pierce/Thermo Fisher, Waltham, MA). For trypsinization, 100 μg of protein (when used for 4-plex iTRAQ labeling, experiment A) or 50 μg (when used for 8-plex iTRAQ labeling, experiment B) was resuspended in 20 μl of 500 mm TEAB, 2% acetonitrile plus 0.08% SDS. Reduction of disulfide bonds with Tris(2-carboxyethyl) phosphine hydrochloride, cysteine-modification with methyl-methanethiosulfonate (MMTS) were performed according to the manufacturer's protocol for iTRAQ (Applied Biosystems, Foster City, CA). For enzymatic digestion, trypsin gold (Cat.: V5280, Promega) was reconstituted in 500 mm TEAB and 5 mm calcium chloride, and used in 1:6 (μg/μg) trypsin-to-protein ratio. Digestion was performed over night at 37 °C. Undigested material was spun down for 10 min at 14,000 × g. The pellets were suspended in TEAB/acetonitrile/SDS solution as before and digested for 5 h at 37 °C with 0.8 μg trypsin per sample. The corresponding samples from two digests were combined, freeze-dried and suspended in 15 μl 500 mm TEAB. The 8-plex iTRAQ labeling was performed according to the manufacturer's protocol with a few modifications. Each label was reconstituted in 210 μl 100% isopropanol and to each sample of 15 μl, 100 μl reconstituted label was added, so that each label was used for two samples. The four-plex iTRAQ labeling was performed according to the manufacturer's protocol except that each label was resuspended in 200 μl ethanol and combined with 20 μl tryptic digest. The samples were incubated for at least 2.5 h at room temperature and stored at –20 until required. Organic solvent (isopropanol or ethanol) was removed by evaporation. Each sample was suspended in 100 μl water. From each sample, 50 μl were combined (200 μg) and concentrated to a volume of 250 μl. The same volume of twofold concentrated SCX buffer A (see below) was added, the pH was adjusted to 2.7 with phosphoric acid. The peptide mixture was subjected to chromatography and mass spectrometry analysis. For off-line peptide pre-fractionation, a silica-based Polysulfoethyl Aspartamide SCX column was used (Cat.: 202SE0502 PolyLC Inc., Columbia USA). The column was run at a flow rate of 200 μl/min on an AKTA purifier (GE Healthcare). Gradient solutions A: 10 mm triethylammonium phosphate, pH 2.7, 25% acetonitrile; B: 10 mm triethylammonium phosphate, pH 2.7, 25% acetonitrile, 500 mm KCl. Gradient conditions: column equilibration with five column volumes (CV) (1 CV = 0.7 ml) of 100% A. Peptides were loaded in 100% A and the column was washed with 10 CV at 100% A. Peptides were eluted: 1) 0 to 5% B in 5 CV; 2) followed by 12 to 30% B in 10 CV; and 3) 24–60% B in 5 CV. Fractions of elution steps 1 and 2 were collected every 45 s, and fractions of the elution step 3 were collected every 1 min in a 96-well plate. Eluted peptides were dried in a vacuum centrifuge and resuspended in 50 μl of 0.1% TFA. Depending on the complexity, either separate fractions or pools of two fractions were analyzed by RP-LC MALDI-time-of-flight (TOF)/TOF. Peptides were trapped on a pre-column (300 μm x 5 mm, C18 PepMap300, LC Packing) and then separated on a C18 capillary column (C18 PepMap 300, 75 μm × 150 mm, 3 μm particle size, LC-Packing) mounted on the Dionex Ultraflex 3000 LC system (LC Packings, Amsterdam, The Netherlands). Mobile phase solutions contained A: 0.05% TFA; B: 0.05% TFA, 80% acetonitrile. Gradient conditions: equilibration of column, binding and washing of peptides was performed with 3% B, elution with 3 to 50% B in 60 min at a flow rate of 300 nL/min. The eluting peptides were mixed 1:4 with 2.2 mg/ml α-cyano-4-hydroxycinnamic acid matrix (LaserBio Labs, Sophia-Antipolis, France) and spotted directly onto a MALDI target (12 s x 260 spots), using a Probot system (LC Packings, Amsterdam, The Netherlands). Peptides were analyzed with a 4800 Proteomics analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems). The MALDI-TOF/TOF was operated in reflectron positive ionization mode in the m/z range 900–4000. The 15 most intense peaks above the signal-to-noise threshold of 120 from each MS spectrum of odd-numbered RP-LC runs were selected for MS/MS fragmentation in the m/z range from 900 to 2000. The 10 most intense peaks above the signal-to-noise of 50 were selected from each MS spectrum of even-numbered RP-LC runs in the m/z range from 2000 to 4000. The MS/MS spectra were acquired using 2 kV acceleration voltage and air as collision gas at 5 × 10−7 Torr. The precursor mass transmission window was set to 300 (full width at half maximum, FWHM) for peptides in the m/z range of 900–2000, and to 200 (FWHM) in the range of 2000–4000 m/z. The peak-lists of the acquired MS/MS spectra were generated, using default settings and the S/N threshold of 10. The MS spectra were calibrated in the plate model mode, using 4700 calibration mixture (Applied Biosystems). MS/MS calibration of the instrument was performed when required, using ACTH 18–39 (m/z = 2465.199) fragment ions. MS/MS peak-lists were extracted by the ProteinPilot software, version 2.0, using default parameters and were automatically submitted to a database search. All MS/MS spectra were analyzed using Mascot (Matrix Science, London, UK; version 2.0) and X!Tandem (www.thegpm.org; version 2007.01.01.1). Mascot and X!Tandem were set up to search a combined L. lactis sp. cremoris MG1363 database, allowing one missed cleavage of the digestion by trypsin. The database was created by combining forward and reversed entries of the L. lactis proteome (release version 31.08.07) and included sequences of porcine trypsin (NCBI accession: P00761), human keratins (P35908, P35527, P13645, NP_006112), chloramphenicol acetyltransferase (P00485), replication protein A (Q04138), and the human CFTR (NCBI accession: NP_000483) containing in total 4902 protein entries. Mascot and X!Tandem searches were performed with a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 200 ppm. MMTS modification of cysteine and Applied Biosystems 4-plex or 8-plexed iTRAQ quantitation chemistry of lysine and the N terminus were specified in Mascot and X!Tandem as fixed modifications. Deamidation of asparagine and glutamine, oxidation of methionine and Applied Biosystems 4-plex or 8-plexed iTRAQ quantitation chemistry of tyrosine were specified in Mascot and X!Tandem as variable modifications. Scaffold (version Scaffold-2_02_03, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (22Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3912) Google Scholar). Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 uniquely identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (23Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3655) Google Scholar). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principle of parsimony. Those peptides were removed from the dataset when quantification was performed. The false positive rate was calculated by dividing 2 times the number of proteins identified in the reversed database by 4902, the sum of all proteins identified in forward and reversed versions of the database. In all measured samples, no hits from the reversed database were detected, using the criteria described above. The relative quantification was based on peptides that were chemically labeled with isobaric reagents, using the 4-plex or 8-plex iTRAQ technique. The quantification information was obtained from the peak areas of the reporter ions (m/z 112.2, 113.2, 114.2, 115.2, 116.2, 117.2, 118.2, 119.2, and 121.2). The peak areas were extracted from the MS/MS spectra by the ProteinPilot software using default settings as specified by the ProteinPilot for the 4800 MALDI instruments (Applied Biosystems). The peak areas were corrected for isotopic impurities by the ProteinPilot using the information provided by the manufacturer in the Certificate of Analysis for each iTRAQ batch. To select quantification data, those ratios were removed where the peak area of one reporter ion was below the signal-to-noise threshold of 10. The global bias correction was performed for all identified peptides. The bias correction factor for a given iTRAQ ratio (e.g. 113/114) was calculated as the sum of all reporter peak areas in all measured spectra from one iTRAQ reagent (e.g. 114) divided by the sum of reporter peak areas of another reagent (e.g. 113). To obtain the bias-corrected peptide iTRAQ ratios, all measured ratios (in this example all 113/114 ratios) were multiplied by the correction factor. The bias-corrected peptide ratios of the same protein were weight-averaged and protein iTRAQ ratios were obtained according to the method used by the ProteinPilot software (Applied Biosystems). Peptides that matched to multiple proteins were excluded from quantification. The relevant protein and peptide data and given in supplemental Tables S5 and S6. To identify proteins with significantly changed abundances two different methods were used depending on the number of available replicate values. Rank Sum analysis was used for the comparison of the CFTR expressing strain versus the control strain at the 4 h time point, where four independent replicates were available. Rank Sum is a nonparametric statistical method based on the Rank Product analysis (24Breitling R. Armengaud P. Amtmann A. Herzyk P. Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments.FEBS Letts. 2004; 573: 83-92Crossref PubMed Scopus (1222) Google Scholar, 25Breitling R. Herzyk P. Rank-based methods as a non-parametric alternative of the T-statistic for the analysis of biological microarray data.J. Bioinform. Comput. Biol. 2005; 3: 1171-1189Crossref PubMed Scopus (116) Google Scholar), which allows the data from biological replicates to be analyzed in a robust way. For the Rank Sum analysis the weighted protein ratios for each of the four replicate samples were calculated as described above and sorted in descen
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