Experimental procedures for proteome analysis of the genome of Aspergillus oryzae RIB40 (= NBRC 100959)
Method
Strain and Culture
A. oryzae RIB40 was grown in YPD media (liquid culture or Membrane-transfer culture(1)).
(1)Ishida H. et al. (1998) Regulation of the Glucoamylase-Encoding Gene (glaB), Expressed in Solid-State Culture (Koji) of Aspergillus oryzae, J. Ferment. Bioeng. 86, 301-307.
A. oryzae RIB40 was grown in YPD media (liquid culture or Membrane-transfer culture(1)).
(1)Ishida H. et al. (1998) Regulation of the Glucoamylase-Encoding Gene (glaB), Expressed in Solid-State Culture (Koji) of Aspergillus oryzae, J. Ferment. Bioeng. 86, 301-307.
Identification methods
2D-PAGE
Protein preparation and separation - Cells were disrupted on dry ice and extracted by acetone precipitation. Extracted proteins were suspended in an extraction buffer (7M Urea, 2M Thiourea, 40mM Tris, 50mM DTT, 4% CHAPS, 0.2% IPG buffer) at room temperature for 1 h, followed by centrifugation to remove debris. IEF was performed IPG strips pH3-10 (GE Healthcare) or pH3-6 or pH5-8 (Bio-Rad). Protein samples were dissolved in 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 40 mM Tris, and 0.2% IPG buffer, and incubated at room temperature for 1 h. IEF was performed using a IPGphor (GE Healthcare) at 20¡ëC (200 V for 2 h and linear gradient of 200-4,000 V for 8 h and finally at 8,000 V to achieve a total of 60 kV-h). After IEF, the strips were equilibrated for 30 min in 6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl (pH 6.8), 1% DTT. SDS-PAGE was then carried out on 12 or 16% gels. Proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Nacalai).
Enzymatic Digestion - In-gel digestion with modified trypsin (sequencing grade, Promega) and sample spotting for MALDI-TOF MS were performed with the Investigator ProPrep automatic digestion system (Genomic Solutions) according to the manufacturer's protocols with some modifications. The CBB-stained protein spots were excised from the gel and washed with 25 mM NH4HCO3 and acetonitrile at room temperature. The proteins were reduced with 10 mM DTT in 25 mM NH4HCO3 at 60¡ëC for 10 min and alkylated with 40 mM iodoacetamide in 25 mM NH4HCO3 at room temperature for 35 min. The dried gel pieces were rehydrated and incubated in 25 mM NH4HCO3 containing modified trypsin at 37¡ëC for 4 h. 3%formic acid was added to stop the enzymatic reaction, and the resultant peptides were concentrated, desalted by passing through a µ-C18 ZipTip (Millipore), mixed with a matrix solution of 50% acetonitrile saturated with ¦Á-cyano-4-hydroxycinnamic acid (Sigma), and air dried on the target plate.
MALDI-TOF MS - The resulting peptide mixture was subjected to analysis on an Ultraflex instrument (Bruker Daltonics). For peptide assignment the mass spectrum data were analyzed using the MASCOT database search program (Matrix Science Ltd.) in the peptide mass fingerprinting mode against the database of putative proteins of A. oryzae predicted ORFs as well as against the translations of the entire genomic sequence in all 6 frames.
Enzymatic Digestion - In-gel digestion with modified trypsin (sequencing grade, Promega) and sample spotting for MALDI-TOF MS were performed with the Investigator ProPrep automatic digestion system (Genomic Solutions) according to the manufacturer's protocols with some modifications. The CBB-stained protein spots were excised from the gel and washed with 25 mM NH4HCO3 and acetonitrile at room temperature. The proteins were reduced with 10 mM DTT in 25 mM NH4HCO3 at 60¡ëC for 10 min and alkylated with 40 mM iodoacetamide in 25 mM NH4HCO3 at room temperature for 35 min. The dried gel pieces were rehydrated and incubated in 25 mM NH4HCO3 containing modified trypsin at 37¡ëC for 4 h. 3%formic acid was added to stop the enzymatic reaction, and the resultant peptides were concentrated, desalted by passing through a µ-C18 ZipTip (Millipore), mixed with a matrix solution of 50% acetonitrile saturated with ¦Á-cyano-4-hydroxycinnamic acid (Sigma), and air dried on the target plate.
MALDI-TOF MS - The resulting peptide mixture was subjected to analysis on an Ultraflex instrument (Bruker Daltonics). For peptide assignment the mass spectrum data were analyzed using the MASCOT database search program (Matrix Science Ltd.) in the peptide mass fingerprinting mode against the database of putative proteins of A. oryzae predicted ORFs as well as against the translations of the entire genomic sequence in all 6 frames.
1D-PAGE
Protein preparetion and separation - Cells were disrupted on dry ice and extracted by acetone precipitation. Extracted proteins were partly digested with glycopeptidase F (TaKaRa) to remove sugar chains.Protein samples were dissolved in 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 40 mM Tris and incubated at room temperature for 1 h. Subsequently a 6 x electrophoresis loading buffer containing 0.35 M Tris-HCl (pH 6.8), 10% SDS, 30% glycerol, and 9.3% DTT was added. 1D-SDS-PAGE was performed on 12% or 16% gels.
Enzymatic Digestion - After CBB staining, the 12% gels were sliced into 30 pieces from the top to the bottom and the 16% gels were sliced into 13 pieces from the 22kDa band to the bottom line (4.4 kDa). In-gel digestion with modified trypsin was performed using Investigator ProPrep for 4 h and stopped with 3% formic acid. The resulting peptide mixtures were eluted from the gel and dried by evaporation. The peptides were diluted with 0.02% formic acid containing 0.005% heptafluorobutyric acid (HFBA) and 2% acetonitrile.
LC/MS/MS - To analyze peptides, LC was combined with nano-ESI-MS/MS. The analysis was performed with a Finnigan LTQ ion trap mass spectrometer (Thermo Electron) coupled with an LC (Michrom Bioresources) through a nanoelectrospray ion source (AMR Inc). The system was fitted with a Peptide CapTrap (Michrom Bioresources) for desalting and concentration, a C18 reverse phase column (50-mm length and 0.2-mm inner diameter; Michrom Bioresources) and a Pico Tip (New Objective) as the electrosprayer. Desalting was performed with a mixture of 0.1% trifluoroacetic acid, 2% acetonitrile, and 98% water. For reverse phase chromatography, Buffer A (0.1% formic acid, 0.005% HFBA, 2% acetonitrile, and 98% water) and Buffer B (0.1% formic acid, 0.005% HFBA, 90% acetonitrile, and 10% water) were used to form a two-step gradient of 5-65% of Buffer B in 50 min at a flow rate of 1 µl/min. The mass spectrometer was operated in data-dependent MS/MS mode with dynamic exclusion at 450 2000 m/z ranges, and the ions were selected for CID with automatic data-dependent settings. The MS/MS spectra were searched for with the MASCOT database search program in the MS/MS mode against the A. oryzae genomic data. The criteria adopted for protein identification were that at least one peptide with ion score 59 or higher matches.
Enzymatic Digestion - After CBB staining, the 12% gels were sliced into 30 pieces from the top to the bottom and the 16% gels were sliced into 13 pieces from the 22kDa band to the bottom line (4.4 kDa). In-gel digestion with modified trypsin was performed using Investigator ProPrep for 4 h and stopped with 3% formic acid. The resulting peptide mixtures were eluted from the gel and dried by evaporation. The peptides were diluted with 0.02% formic acid containing 0.005% heptafluorobutyric acid (HFBA) and 2% acetonitrile.
LC/MS/MS - To analyze peptides, LC was combined with nano-ESI-MS/MS. The analysis was performed with a Finnigan LTQ ion trap mass spectrometer (Thermo Electron) coupled with an LC (Michrom Bioresources) through a nanoelectrospray ion source (AMR Inc). The system was fitted with a Peptide CapTrap (Michrom Bioresources) for desalting and concentration, a C18 reverse phase column (50-mm length and 0.2-mm inner diameter; Michrom Bioresources) and a Pico Tip (New Objective) as the electrosprayer. Desalting was performed with a mixture of 0.1% trifluoroacetic acid, 2% acetonitrile, and 98% water. For reverse phase chromatography, Buffer A (0.1% formic acid, 0.005% HFBA, 2% acetonitrile, and 98% water) and Buffer B (0.1% formic acid, 0.005% HFBA, 90% acetonitrile, and 10% water) were used to form a two-step gradient of 5-65% of Buffer B in 50 min at a flow rate of 1 µl/min. The mass spectrometer was operated in data-dependent MS/MS mode with dynamic exclusion at 450 2000 m/z ranges, and the ions were selected for CID with automatic data-dependent settings. The MS/MS spectra were searched for with the MASCOT database search program in the MS/MS mode against the A. oryzae genomic data. The criteria adopted for protein identification were that at least one peptide with ion score 59 or higher matches.
MDLC
Protein Separation - Proteins of a whole cell lysate were separated by off-line 2D-LC. The first dimensional chromatography was performed with a self-packed strong anion exchange (SAX) column prepared in a glass chromatography tube of 15-mm inner diameter and 300-mm length. Trimethylaminopropyl-bonded silica gel (BONDESIL-SAX, 40 µm, Varian) was used to fill the column. Buffers used were 20 mM Tris-HCl, pH 7.0 (Buffer C), and 20 mM Tris-HCl, pH 7.0, with 1 M NaCl (Buffer D). Proteins were eluted with Buffer C from 0 to 5 min with a linear gradient of BufferC to Buffer D from 6 to 20 min and with Buffer D from 21 to 30 min. The flow rate was 10 ml/min, and the eluate was collected into four 50-ml fractions. The fractions were concentrated to 2ml by partial lyophilization (EYELA FD-81, Tokyo Rikakikai). The second dimensional chromatography was performed with a gel permeation chromatography (GPC) column (Bioassist G2SWXL, TOSOH). 200 µl of each of the concentrated SAX fractions were successively injected into a column connected with a guard column (TOSOH) and two GPC. Elution was performed with Buffer E (0.1 M sodium phosphate, pH 7.0) at a flow rate of 0.5 ml/min, and fractions were collected every 3.5 min starting from 18 min until 60 min (6 fractions). In this way a total of 24 fractions (4 x 6) were obtained.
In-column Enzymatic Digestion - Samples in each fraction were reduced by incubating in 5 mM DTT at 60 ¡ëC for 30 min, alkylated with 15 mM iodoacetamide in the dark and at room temperature for 30 min, and digested with modified trypsin at 37 ¡ëC for 12h. The samples were adjusted to pH 4 with trifluoroacetic acid, desalted with a C18 reverse phase column, and evaporated. The digests were dissolved in a mixture of 0.02% formic acid, 0.005% HFBA, 2% acetonitrile, and 98% water prior to MS analysis.Proteins retained on the SAX column were treated with 0.1% RapiGest (Waters) in 5 mM DTT and incubated at 60 ¡ëC for 30 min. The proteins were alkylated and digested with modified trypsin at 37 ¡ëC for 15 h. The digests were eluted with a mixture of 0.1% trifluoroacetic acid, 5% methanol, and 94.9% water; desalted with a C18 reverse phase column; and evaporated. The digests were dissolved in 0.02% formic acid with 0.005% HFBA, 2% acetonitrile, and 98% water prior to MS analysis.
LC/MS/MS - To analyze peptides, LC/MS/MS were performed as described in the previous section <1D-PAGE-LC>.
In-column Enzymatic Digestion - Samples in each fraction were reduced by incubating in 5 mM DTT at 60 ¡ëC for 30 min, alkylated with 15 mM iodoacetamide in the dark and at room temperature for 30 min, and digested with modified trypsin at 37 ¡ëC for 12h. The samples were adjusted to pH 4 with trifluoroacetic acid, desalted with a C18 reverse phase column, and evaporated. The digests were dissolved in a mixture of 0.02% formic acid, 0.005% HFBA, 2% acetonitrile, and 98% water prior to MS analysis.Proteins retained on the SAX column were treated with 0.1% RapiGest (Waters) in 5 mM DTT and incubated at 60 ¡ëC for 30 min. The proteins were alkylated and digested with modified trypsin at 37 ¡ëC for 15 h. The digests were eluted with a mixture of 0.1% trifluoroacetic acid, 5% methanol, and 94.9% water; desalted with a C18 reverse phase column; and evaporated. The digests were dissolved in 0.02% formic acid with 0.005% HFBA, 2% acetonitrile, and 98% water prior to MS analysis.
LC/MS/MS - To analyze peptides, LC/MS/MS were performed as described in the previous section <1D-PAGE-LC>.