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• W2483577433 abstract "Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. The field of recombinant protein production is constantly growing, providing both bulk scale enzymes for industrial processes as well as tailor-made proteins, such as antibody fragments, for medical usage. Yeasts have become a well utilized platform for recombinant protein production thanks to their ability to secrete proteins of interest (1.Mattanovich D. Branduardi P. Dato L. Gasser B. Sauer M. Porro D. Recombinant protein production in yeasts.Methods Mol. Biol. 2012; 824: 329-358Crossref PubMed Scopus (222) Google Scholar, 2.Mattanovich D. Sauer M. Gasser B. Yeast biotechnology: teaching the old dog new tricks.Microb. Cell Fact. 2014; 13: 34Crossref PubMed Scopus (67) Google Scholar). This greatly facilitates product purification and thus helps to make a process economically competitive (3.Idiris A. Tohda H. Sasaki M. Okada K. Kumagai H. Giga-Hama Y. Takegawa K. Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway.Appl. Microbiol. Biotechnol. 2010; 85: 667-677Crossref PubMed Scopus (55) Google Scholar). As unicellular microorganisms they are easy to cultivate and high cell densities can be reached for industrial production processes. As eukaryotes they possess both protein folding machinery and the ability to perform post-translational modifications related to higher eukaryotes, which can be advantageous compared with prokaryotic microorganisms (1.Mattanovich D. Branduardi P. Dato L. Gasser B. Sauer M. Porro D. Recombinant protein production in yeasts.Methods Mol. Biol. 2012; 824: 329-358Crossref PubMed Scopus (222) Google Scholar). Additionally, a large amount of research has been performed over the past decades into improving the secretion of recombinant proteins from yeast cells, engineering intracellular transport of secreted proteins as well as glycosylation patterns and protein folding. Recent studies also deal with the complex interactions between metabolism and recombinant protein production and systems biology tools allow the identification and overcoming of metabolic bottlenecks in recombinant protein production and secretion (4.Hou J. Tyo K.E. Liu Z. Petranovic D. Nielsen J. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae.FEMS Yeast Res. 2012; 12: 491-510Crossref PubMed Scopus (141) Google Scholar, 5.Klein T. Niklas J. Heinzle E. Engineering the supply chain for protein production/secretion in yeasts and mammalian cells.J. Ind. Microbiol. Biotechnol. 2015; 42: 453-464Crossref PubMed Scopus (22) Google Scholar). Although Saccharomyces cerevisiae and Pichia pastoris are by far the most commonly utilized yeasts for industrial production processes (6.Porro D. Gasser B. Fossati T. Maurer M. Branduardi P. Sauer M. Mattanovich D. Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems?.Appl. Microbiol. Biotechnol. 2011; 89: 939-948Crossref PubMed Scopus (76) Google Scholar, 7.Damasceno L.M. Huang C.J. Batt C.A. Protein secretion in Pichia pastoris and advances in protein production.Appl. Microbiol. Biotechnol. 2012; 93: 31-39Crossref PubMed Scopus (220) Google Scholar), alternative host systems should also be considered. The fission yeast Schizosaccharomyces pombe is one such alternative cell factory. Although this yeast is well known as a model organism for molecular and cell biology, its application for recombinant protein production has been demonstrated previously (8.Takegawa K. Tohda H. Sasaki M. Idiris A. Ohashi T. Mukaiyama H. Giga-Hama Y. Kumagai H. Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system.Biotechnol. Appl. Biochem. 2009; 53: 227-235Crossref PubMed Scopus (51) Google Scholar). In addition to having a well sequenced and annotated genome for which data is freely available (www.pombase.org), S. pombe is very well characterized in respect to cell cycle regulation, DNA replication, transcription and translation as well as in terms of protein folding and protein quality control (9.Celik E. Calik P. Production of recombinant proteins by yeast cells.Biotechnol. Adv. 2012; 30: 1108-1118Crossref PubMed Scopus (227) Google Scholar). Post-translational modification of secreted proteins, especially glycosylation, is closely related to mammalian cells (10.Parodi A.J. Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells.Biochim. Biophys. Acta. 1999; 1426: 287-295Crossref PubMed Scopus (93) Google Scholar), making S. pombe an attractive host for recombinant mammalian protein production. Secretion of recombinant human transferrin has been reported, and single-chain antibody fragments have been produced with titers up to 5 mg/L (11.Mukaiyama H. Giga-Hama Y. Tohda H. Takegawa K. Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.Appl. Microbiol. Biotechnol. 2009; 85: 155-164Crossref PubMed Scopus (21) Google Scholar, 12.Naumann J.M. Kuttner G. Bureik M. Expression and secretion of a CB4–1 scFv-GFP fusion protein by fission yeast.Appl. Biochem. Biotechnol. 2011; 163: 80-89Crossref PubMed Scopus (17) Google Scholar). In recent times a significant amount of effort has been focused toward engineering S. pombe as a competitive host system for recombinant protein secretion (3.Idiris A. Tohda H. Sasaki M. Okada K. Kumagai H. Giga-Hama Y. Takegawa K. Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway.Appl. Microbiol. Biotechnol. 2010; 85: 667-677Crossref PubMed Scopus (55) Google Scholar, 11.Mukaiyama H. Giga-Hama Y. Tohda H. Takegawa K. Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.Appl. Microbiol. Biotechnol. 2009; 85: 155-164Crossref PubMed Scopus (21) Google Scholar). Despite these efforts, S. pombe is still underdeveloped as an industrial cell factory and further research will be necessary to create a host system competitive to S. cerevisiae and P. pastoris. In this study, we performed a quantitative (comparative) proteome analysis on three different strains of S. pombe secreting the model protein maltase in varying amounts: the wild type strain (NW8), a moderate maltase producing strain (NW9), and a strong maltase producing strain (NW10). In a recent study, we investigated the effects of increased maltase secretion on the central carbon metabolism of S. pombe using 13C-assisted metabolic flux analysis (13.Klein T. Lange S. Wilhelm N. Bureik M. Yang T.H. Heinzle E. Schneider K. Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe–a quantitative approach using 13C-based metabolic flux analysis.Metab. Eng. 2014; 21: 34-45Crossref PubMed Scopus (33) Google Scholar). In this study, we were interested in the system wide proteomic response of S. pombe cells to the increased level of protein secretion. Our model protein maltase, with an approximate size of 110 kDa, is a highly secreted α-glucosidase of S. pombe (14.Jansen M.L. Krook D.J. De Graaf K. van Dijken J.P. Pronk J.T. de Winde J.H. Physiological characterization and fed-batch production of an extracellular maltase of Schizosaccharomyces pombe CBS 356.FEMS Yeast Res. 2006; 6: 888-901Crossref PubMed Scopus (16) Google Scholar). It is encoded by the agl1 gene and expression of the wildtype gene is tightly regulated by extracellular glucose levels. Although a strong secretion up to 10 mg (g cell dry weight (CDW)−1) 1The abbreviations used are: CDWcell dry weightFDRfalse discovery rateGISglobal internal standardGOgene ontologyIP-RPion pairing reversed phasePPPpentose phosphate pathwayTCAtricarboxylic acid cycleμgrowth rate2D-LCtwo-dimensional liquid chromatography. takes place under glucose starvation, high glucose concentrations in the media suppress gene expression (14.Jansen M.L. Krook D.J. De Graaf K. van Dijken J.P. Pronk J.T. de Winde J.H. Physiological characterization and fed-batch production of an extracellular maltase of Schizosaccharomyces pombe CBS 356.FEMS Yeast Res. 2006; 6: 888-901Crossref PubMed Scopus (16) Google Scholar). We expressed the agl1 gene under the control of the strong nmt1 promoter to achieve constitutive expression by plasmid as well as via additional chromosomal integration, which led to increased amounts of secreted protein even when high amounts of glucose are present in the media. Proteome analysis was performed using a two dimensional LC coupled offline to MALDI MS. To reduce measurements time, a pooling scheme for the multidimensional separation was established. Quantitative information was generated by isobaric labeling using the iTRAQ approach (15.Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar). A global internal standard approach was applied. cell dry weight false discovery rate global internal standard gene ontology ion pairing reversed phase pentose phosphate pathway tricarboxylic acid cycle growth rate two-dimensional liquid chromatography. The quantitative proteomics results revealed changes in protein levels across numerous biological pathways within the cell. This finding provides a broad set of targets for further genetic engineering and media design with the aim of improved protein secretion. We selected two of these targets, amino acid biosynthesis and membrane fluidity, which could be influenced via supplementation of the media with specific compounds in order to validate the efficacy of the proteome data for increasing protein secretion in S. pombe. Construction of S. pombe strains NW8 and NW9 has been described before (13.Klein T. Lange S. Wilhelm N. Bureik M. Yang T.H. Heinzle E. Schneider K. Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe–a quantitative approach using 13C-based metabolic flux analysis.Metab. Eng. 2014; 21: 34-45Crossref PubMed Scopus (33) Google Scholar). For the construction of strain NW10, the fission yeast gene agl1 (16.Okuyama M. Okuno A. Shimizu N. Mori H. Kimura A. Chiba S. Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe.Eur. J. Biochem. 2001; 268: 2270-2280Crossref PubMed Scopus (66) Google Scholar) was amplified from genomic fission yeast DNA by PCR with flanking primers that introduce restriction sites for NdeI (5′) and BamHI (3′), respectively. After restriction digest the amplified agl1 gene was cloned into the integrative expression vector pCAD1 (17.Dragan C.A. Zearo S. Hannemann F. Bernhardt R. Bureik M. Efficient conversion of 11-deoxycortisol to cortisol (hydrocortisone) by recombinant fission yeast Schizosaccharomyces pombe.FEMS Yeast Res. 2005; 5: 621-625Crossref PubMed Scopus (56) Google Scholar) to yield the new plasmid pCAD1-agl1 and the episomal expression vector pREP1 to yield pREP1-agl1. The correctness of all expression constructs was confirmed by sequencing. The parental strain NCYC 2036 (18.Losson R. Lacroute F. Plasmids carrying the yeast OMP decarboxylase structural and regulatory genes: transcription regulation in a foreign environment.Cell. 1983; 32: 371-377Abstract Full Text PDF PubMed Scopus (115) Google Scholar) was transformed first with the integrative vector pCAD1-agl1 and after successful integration transformed with plasmid pREP1-agl1 using cryocompetent cells (19.Suga M. Hatakeyama T. High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation.Yeast. 2001; 18: 1015-1021Crossref PubMed Scopus (72) Google Scholar). The resulting strain NW10 carries a genomic copy of the agl1 gene integrated into the leu1 locus and a second agl1 gene on the episomal plasmid pREP1-agl1, both under control of the strong nmt1 promoter. All strains are listed in supplemental Table S1. Minimal media consisting of [g/liter] glucose 20.0; NH4SO4 15.0; KH2PO4 11.0; MgCl2 1.0; NaCl 1.0; CaCl2 0.014 was used for cultivations. Vitamins and minerals were added to the following final concentrations (mg/liter): calcium pantothenate 1.0; nicotinic acid 10.0; myo-inositol 10.0; pyridoxine 0.5; biotin 0.01; FeSO4 0.7; ZnSO4 0.8; MnSO4 0.8; boric acid 1.0; CoCl2 1.0; NaMoO4 5.0; KI 2.0; CuSO4 0.08. The pH of the media was set to 5.5. For cultivation with addition of amino acids, 5 mm of the following amino acids were added: asparagine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, tryptophan and valine. Media components, amino acids and fluconazole were purchased from Sigma-Aldrich (St. Louis, MO). Fluconazole was applied in cultivations at concentrations ranging from 0.5 to 10 μg/ml. Cell growth was observed via optical density measurement at 595 nm. Correlation between optical density at 595 nm (OD595) and cell dry weight was taken from Klein et al. (20.Klein T. Schneider K. Heinzle E. A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off-gas.Biotechnol. Bioeng. 2013; 110: 535-542Crossref PubMed Scopus (32) Google Scholar). For analysis of culture supernatants, 1 ml of cells was harvested in regular intervals during the growth phase on glucose and supernatants were analyzed by HPLC as described before (20.Klein T. Schneider K. Heinzle E. A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off-gas.Biotechnol. Bioeng. 2013; 110: 535-542Crossref PubMed Scopus (32) Google Scholar). Ethanol was determined enzymatically (Ethanol UV method, product code 10176290035, R-Biopharm, Darmstadt, Germany). For determination of the average amino acid composition of cellular protein, 1 ml of cells were harvested during the glucose growth phase, the pellet resuspended in 100 μl of 6 m HCl and the cellular protein hydrolyzed for 24 h at 100 °C. The suspension was neutralized by addition of NaOH, the cell debris removed by filtration through a 0.22 μm filtration unit, and the remaining solution lyophilized. The lyophilizate was resuspended in 500 μl of 200 μm α-butyric acid, which served as internal standard for HPLC analysis, which was performed as described before (21.Bolten C.J. Wittmann C. Appropriate sampling for intracellular amino acid analysis in five phylogenetically different yeasts.Biotechnol. Lett. 2008; 30: 1993-2000Crossref PubMed Scopus (52) Google Scholar). Ergosterol was extracted from dry cell mass as described for Saccharomyces cerevisiae (22.Shobayashi M. Mitsueda S. Ago M. Fujii T. Iwashita K. Iefuji H. Effects of culture conditions on ergosterol biosynthesis by Saccharomyces cerevisiae.Biosci. Biotechnol. Biochem. 2005; 69: 2381-2388Crossref PubMed Scopus (59) Google Scholar). Quantification was performed by HPLC with 95% methanol as eluent at a flow rate of 1 ml/min and 30 °C oven temperature (22.Shobayashi M. Mitsueda S. Ago M. Fujii T. Iwashita K. Iefuji H. Effects of culture conditions on ergosterol biosynthesis by Saccharomyces cerevisiae.Biosci. Biotechnol. Biochem. 2005; 69: 2381-2388Crossref PubMed Scopus (59) Google Scholar). Ergosterol standards were in the range of 0.1 to 2 mg/ml. All measurements were performed in biological and technical duplicates. Maltase activity assay was performed as described earlier (14.Jansen M.L. Krook D.J. De Graaf K. van Dijken J.P. Pronk J.T. de Winde J.H. Physiological characterization and fed-batch production of an extracellular maltase of Schizosaccharomyces pombe CBS 356.FEMS Yeast Res. 2006; 6: 888-901Crossref PubMed Scopus (16) Google Scholar). Glucose produced from maltase hydrolysis was determined enzymatically (d-Glucose UV method, product code 10716251035, R-Biopharm, Germany). Dithiothreitol (DTT), CHAPS, urea, glass beads, iodoacetamide (IAA), triethylammonium bicarbonate (TEAB), triethylamine, acetonitrile (ACN), trifluoroacetic acid (TFA), formic acid (FA), α-cyano-4-hydroxycinnamic acid (CHCA), and Glu-1-fibrinopeptide B (Glu-Fib) were from Sigma-Aldrich (Taufkirchen, Germany). Protease inhibitor mixture was from Roche (Mannheim, Germany). Trypsin was obtained from Promega (Madison, WI). 4-plex iTRAQ reagent kit was from Applied Biosystems (Darmstadt, Germany). Tris-HCl was from Roth (Karlsruhe, Germany). Water used for all experiments was purified by an arium®611VF System (Sartorius, Göttingen, Germany). Strains of S. pombe grown in 50 ml culture were pelleted by centrifugation, frozen immediately in liquid nitrogen and stored at −80 °C. For preparation of the total cell lysates of S. pombe, 20 ml cells (ca. 2 × 109 cells) were re-suspended in 550 μl of freshly prepared ice-cold denaturing lysis buffer containing protease inhibitors (10 mm Tris-HCl pH 7.4, 0.55% CHAPS, 8 m urea, 200 mm DTT, 5 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mm phenylmethylsulfonylfluoride) (23.Schmidt M.W. Houseman A. Ivanov A.R. Wolf D.A. Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe.Mol. Syst. Biol. 2007; 3: 79Crossref PubMed Scopus (99) Google Scholar) and ca. 200 mg of 0.25–0.5 mm glass beads were added. Cells were disrupted in an oscillating mill device (MM400, Retsch, Haan, Germany) at 30 Hz/s for 120 s, paused for another 120 s on ice, the procedure was repeated five times. Cell homogenate was centrifuged at 14,000 × g for 15 min at 4 °C and the supernatant (clear cell lysate) were transferred to a 2 ml Eppendorf tube. The cell lysate extraction step was repeated twice and the supernatants were combined. The protein concentration was determined by Bradford assay (Bio-Rad Inc., Hercules, CA). Four hundred micrograms cell lysate proteins from each strain of the S. pombe were alkylated with 20 μl of 375 mm IAA at RT for 30 min in the dark before transferring to the 0.5 ml Amicon ultra centrifugal filter unit (MWCO: 3 kDa, Millipore, Billerica, MA) for buffer exchange and concentration. The ultrafiltration microcentrifuge tube was centrifuged at 14,000 × g for 25 min at 4 °C, re-buffered twice with 150 μl of H2O, once with 150 μl of 200 mm TEAB, and the concentrate was recovered by reverse spinning at 1000 × g for 2 min at 4 °C. The protein concentration of the purified cell lysates was determined again by Bradford assay. For trypsin digestion, 2 μg of trypsin was added to 100 μg of extracted cell lysate (adjusted with 0.2 M TEAB to a final volume of 115 μl) and incubated at 37 °C overnight. Digestion was stopped by freezing samples at −20 °C. Isobaric labeling of the resulting peptides was accomplished with 4-plex iTRAQ reagent. Prior to labeling, one quarter of the peptide solution from each of the three samples (i.e. NW8, NW9, and NW10) were combined to create a reference sample (i.e. the global internal standard, Mix), as shown in Fig. 1A. The peptide solutions were concentrated to less than 30 μl in a SpeedVac (Eppendorf Vacuum Concentrator Plus, Wesseling-Berzdorf, Germany) prior to labeling with 4-plex iTRAQ reagents according to the manufacturer's protocol. Peptides derived from the Mix, NW8, NW9, and NW10 were labeled with iTRAQ tags 114, 115, 116, and 117, respectively, at room temperature for 2 h. The four iTRAQ-labeled peptide samples were pooled and stored at −80 °C until MS analysis. Three independent biological replicates were performed. First dimension peptide separation was performed by reversed-phase RP-HPLC at high pH (24.Delmotte N. Lasaosa M. Tholey A. Heinzle E. Huber C.G. Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis.J. Proteome Res. 2007; 6: 4363-4373Crossref PubMed Scopus (116) Google Scholar) on an Ultimate 3000 Binary Analytical HPLC system (Dionex, Dreieich, Germany) using a reversed phase Gemini 3 μm C18 110 Å column (2 mm i.d. × 150 mm, Phenomenex, Aschaffenburg, Germany). The pooled iTRAQ-labeled peptides from 100 μg protein digests were injected. Mobile phase A was 72 mm triethylamine in water (pH 10), and mobile phase B was 72 mm triethylamine in ACN (apparent pH 10). The LC gradient started isocratic with 10% solvent B for 5 min, followed by a linear ramp to 55% solvent B over 30 min; after a second linear ramp to 95% B (in 2 min), this solvent was kept isocratic for an additional 10 min before re-equilibration at 5% B. The flow rate was 200 μl/min; the column temperature was 30 °C; UV detection was performed at 214, 254, and 280 nm. Two-minute wide fractions were collected manually from 7 to 47 min. Every fourth fraction was pooled (i.e. fraction 1 plus 5 plus 9 etc.), yielding a total of four, 10-minute wide fractions. The four pooled fractions were concentrated in a SpeedVac, and re-buffered with 0.1% TFA for second dimension peptide separation. Second dimension peptide separation was performed using ion-pairing-reversed-phase HPLC (IP-RP-HPLC) at pH 2. It was carried out with an integrated Ultimate 3000 RSLCnano system (Dionex) equipped with a μ-precolumn Acclaim PepMap 100 (300 μm i.d. × 5 mm, 100 Å pore and 5 μm particle size, Dionex) and an analytical reversed-phase Acclaim PepMap 100 C18 column (75 μm i.d. × 250 mm, 100 Å pore and 3 μm particle size, Dionex) that was coupled online to a Probot microfraction collector (LC Packings, Amsterdam, the Netherlands) for MALDI target spotting. Mobile phase A was 0.05% TFA (pH 2), mobile phase B was 0.04% TFA in 80% ACN (pH 2). The LC gradient was held at initial conditions of 15% B for 5 min followed by a linear ramp to 50% over 120 min; 95% B was reached via a linear gradient over the next 2 min and isocratically held for an additional 10 min before re-equilibration at 10% B. The flow rate was 300 nL/min; the column temperature was 30 °C; UV detection at 214 nm. Eluting peptides were directly mixed in the Probot with matrix solution (3 mg/ml CHCA, 0.1% TFA and 5 nm Glu-Fib in 70% ACN) in a ratio of 1:3 (v/v) and spotted in 15 s intervals. Three technical (injection) replicates from each of the four fractions of first dimension separation were performed. All MS and MS/MS measurements were performed on an AB Sciex TOF/TOFTM 5800 (Darmstadt, Germany) mass spectrometer operated in reflector positive ion mode. MS data were acquired with a total of 1200 shots per spectrum (200 shots per subspectrum) with a laser pulse rate of 400 Hz. MS spectra in LC-MALDI experiments were internally calibrated on Glu-Fib and the matrix cluster signal at m/z of 877.034. MS/MS data were acquired at 1000 Hz in 1 kV MS/MS mode with 4080 shots/spectrum (255 shots per subspectrum). Nonredundant and fully automated precursor ions were selected by the TOF/TOF Series Explorer software (AB Sciex) for MS/MS fragmentation. Precursor ions were separated by timed ion selection with a resolution window of 200 (FHWM), for each spot the 20 most intense precursors with a minimum S/N ratio of 30 were selected for MS/MS, using ambient air as collision gas with gas pressure of 4.0 × 10−6 mbar. Protein identification was performed with the ProteinPilot v4.0 software (AB Sciex) using the Paragon algorithms (25.Shilov I.V. Seymour S.L. Patel A.A. Loboda A. Tang W.H. Keating S.P. Hunter C.L. Nuwaysir L.M. Schaeffer D.A. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.Mol. Cell. Proteomics. 2007; 6: 1638-1655Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar). MS/MS data from 36 HPLC runs (i.e. 3 biological replicates × four pooled fractions from first dimension separation × three technical replicates) were searched separately against the PomBase database (released on 9th of December 2010, 5038 entries) downloaded from EMBL-EBI (ftp://ftp.sanger.ac.uk/pub/yeast/pombe/Protein_data/OLD/Pompep/). The following search parameters were selected: iTRAQ 4-plex peptide label, cysteine carbamidomethylation, trypsin specificity, one missed cleavage site allowed, ID focused on biological modification and amino acid substitution, and processing including quantification and “Thorough ID,” in which the precursor ion mass accuracy and fragment ion mass accuracy are MALDI 5800 built-in function of ProteinPilot software. All reported proteins were identified with 99% confidence as determined by ProteinPilot Unused ProtScores (2.0) with the corresponding local false positive discovery rate (FDR) (26.Tang W.H. Shilov I.V. Seymour S.L. Nonlinear fitting method for determining local false discovery rates from decoy database searches.J. Proteome Res. 2008; 7: 3661-3667Crossref PubMed Scopus (274) Google Scholar) below 5% (supplemental Table S2). The identified proteins were grouped by the ProGroup algorithm (AB Sciex) to minimize redundancy. Protein quantification was performed following extraction of the Peptide Summary data that was obtained from each LC run (a total of 36 spot sets). Only peptides that were confidently identified via the Paragon algorithm and classified as “Used” were exported to Microsoft Excel for further data merging and protein ratio calculation using the in-house VBA (visual basic for applications) scripts (see supplemental Information). These scripts performed steps identical to those of ProteinPilot for the calculation of protein ratios within the three biological replicates; however, they were necessary as with our computational setup ProteinPilot was not able to perform the merging and statistical calculations of all data sets. Only proteins with two or more unique peptides quantified in all three LC-MS experiments were considered for relative quantification, excluding those common to other isoforms or proteins of the same family. A scheme illustrating the iTRAQ ratio comparison strategy used in this study is shown in Fig. 1B. Proteins with significantly different abundances between the conditions were identified by multiple hypotheses testing (Students t test, two tailed assuming equal variance) that was controlled by Benjamini Hochberg FDR analysis (27.Benja" @default.
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• W2483577433 date "2016-10-01" @default.
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• W2483577433 title "Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion" @default.
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