Experimental procedures for proteome analysis of the genome of Aeropyrum pernix K1^T (= NBRC 100138^T)

Strain and Culture
A. pernix strain K1 deposited at NITE Biological Resource Center (NBRC 100138) was cultured at 90 °C in 400 ml of jamarine-yeast extract-trypticase peptone medium for 20 h, cooled down on ice, and harvested by centrifugation at 5,000 xg at 4 °C for 10 min. Cellular pellets were resuspended in 3.5% NaCl and recentrifuged.

Protein Preparation
Two-dimensional(2D)-PAGE and 1D-SDS-PAGE-LC-MS/MS
A. pernix K1 cells were suspended in an extraction buffer (67% acetic acid containing 33 mM MgCl₂) and disrupted by sonication at 4 °C. Cell debris were removed by centrifugation, and 4 volumes of 20 mM DTT in acetone were added to the supernatant. The mixture was stored at -20 °C, and the protein precipitates were collected by centrifugation and dried.
Multidimensional(MD)-LC-MS/MS
A. pernix K1 cells were suspended in distilled water and lysed by homogenization in S-203 (AS ONE, Osaka, Japan) for 30 s on ice.

Identification methods
2D-PAGE
Protein Separation by 2D-PAGE - IEF was performed on either 180-mm IPG strips with the pH range of 3-10 (Amersham Biosciences) or IPG ReadyStrips with the pH range of 3-6 or 5-8 (Bio-Rad). Protein samples were dissolved in a lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 40 mM Tris, and 0.2% carrier ampholyte and incubated at room temperature for 1 h. The first dimensional separation was performed on an IPGphor IEF apparatus (Amersham Biosciences). IPG strips loaded with 100 μg of protein were electrofocused first at 200 V for 1 h, then at a linear gradient of 200-4,000 V for 6 h, and finally at 8,000 V to achieve a total of 60 kV-h. After IEF, the strips were equilibrated with an equilibration buffer containing 6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl (pH 6.8), and 1% DTT for 30 min. SDS-PAGE was then carried out on 12 or 16% polyacrylamide gels (20 x 20 x 0.1 cm). Proteins were visualized by staining with Coomassie Brilliant Blue R-250 (CBB) (Nacalai Tesque, Kyoto, Japan).

Radical-free and Highly Reducing (RFHR)-2D-PAGE - The method of Wada was mainly used. Protein samples were dissolved in a lysis buffer containing 8 M urea and 0.2 M mercaptoethanol and incubated at 40 °C for 30 min. Sample charging electrophoresis was carried out with 100 μg of protein on an 8% polyacrylamide gel containing 8 M urea, 40 mM KOH, and 0.37% acetic acid at 100 V for 30 min on an NA1450 apparatus (Nihon Eido, Tokyo, Japan). Subsequently the first dimensional separation was performed on an 8% polyacrylamide gel containing 8 M urea, 400 mM Tris, 500 mM boric acid, and 21.5 mM EDTA-2Na at 100 V for 15 h on an NA1460 apparatus (Nihon Eido). The second dimensional separation was then carried out on an 18% polyacrylamide gel containing 8 M urea 50 mM KOH and 5% acetic acid (16 x 16 x 0.2 cm) at 100 V for 30 h. Proteins were visualized with CBB as described above.

Enzymatic Digestion for 2D-PAGE-MALDI-TOF MS - In-gel digestion with modified trypsin (sequencing grade, Promega, Madison, WI) and sample spotting for MALDI-TOF MS were performed with the Investigator ProPrep automatic digestion and spotting system (Genomic Solutions, Huntingdon, UK) 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 DDT in 25 mM NH₄HCO₃ at 60 °C 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 °C 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, Billerica, MA), mixed with a matrix solution of 50% acetonitrile saturated with α-cyano-4-hydroxycinnamic acid (Sigma), and airdried on the target plate.


Mass Spectrometry of 2D-PAGE-MALDI-TOF MS - The resulting peptide mixture was subjected to analysis on an Auto-Flex instrument (Bruker Daltonics, Bremen, Germany) with α-cyano-4-hydroxycinnamic acid as the matrix and operated in the reflector mode. Calibration was performed in the external mode using a peptide calibration standard kit (Bruker Daltonics). For peptide assignment the mass spectrum data were analyzed using the MASCOT database search program (Matrix Science Ltd., London, UK) in the peptide mass fingerprinting mode against the database of putative proteins of A. pernix K1 containing the data for 2,694 predicted ORFs as well as against the translations of the entire genomic sequence in all 6 frames.

Amino-terminal Amino Acid Sequence
Protein spots on 2D-PAGE were electroblotted onto a PVDF membrane (Sequi-Blot PVDF membrane, Bio-Rad) with a semidry blotting apparatus (Bio Craft, Tokyo, Japan). The blotted membrane was stained with CBB. Singly stained spots were excised from the PVDF membrane and applied to a protein sequencer (model Procise 491cLC, Applied Biosystems, Foster City, CA) if the staining intensity appeared to be strong enough for sequencing. For weakly stained spots, two to six excised spots were combined by repeating 2DPAGE and then applied to the protein sequencer. Edman reactions were performed according to the manufacturer's instructions. To identify each protein, the amino acid sequences obtained were compared with the predicted amino acid sequence data translated from the genomic sequence of A. pernix K1.

1D-SDS-PAGE-LC-MS/MS
Protein Separation by 1D-SDS-PAGE - Protein samples were dissolved in a lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, and 40 mM Tris and incubated at room temperature for 1 h. Subsequently a 6 x concentrated 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 10% separating polyacrylamide gels with a Tris-Tricine running buffer containing 0.1 M Tris, 0.1 M Tricine, and 0.1% SDS.

Enzymatic Digestion for 1D-SDS-PAGE-LC-MS/MS - After CBB staining, the gels were sliced into 5-mm-thick pieces from the top band (>116 kDa) to the bottom line (4.4 kDa). In-gel digestion with modified trypsin was performed using Investigator ProPrep for 6 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.

Mass Spectrometry of 1D-SDS-PAGE-LC-MS/MS - To analyze peptides, 2D-LC was combined with nano-ESI-MS/MS. The analysis was performed with a Finnigan LCQ DECA XP Plus ion trap mass spectrometer (ThermoElectron, San Jose, CA) coupled with an LC MAGIC 2002 system (Michrom Bioresources, Auburn, CA) through a nanoelectrospray ion source (AMR Inc., Tokyo, Japan). The system was fitted with a strong cation exchange peptide trap column of 1.0-mm inner diameter and 8-mm length (Michrom Bioresources) for the first dimensional chromatography, 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) for the second dimensional chromatography, and a Pico Tip (New Objective, Woburn, MA) as the electrosprayer. The solvents used for strong cation exchange were 0.02% formic acid containing 0.005% HFBA and 2% acetonitrile with either 0, 25, 50, 75, 100, 150, 250, or 500 mM HCOONH₄ (used in eight steps). 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 gradient of 5-65% of Buffer B in 20 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 converted into peak list files with SEQUEST^TM Browser (ThermoElectron) that were searched for with the MASCOT database search program in the MS/MS mode against the A. pernix K1 genomic data. The criteria adopted for protein identification were either 1) that at least three peptides with ion score 20 or higher match or 2) that at least one peptide with ion score 40 or higher matches.

MD-LC-MS/MS
Protein Separation by MD-LC - Proteins of a whole cell lysate were separated by off-line 2D-LC and 2D-LC-nano-ESI-MS/MS. The first dimensional chromatography was performed with a self-packed strong anion exchange (SAX) column prepared in a glass chromatography tube of 8-mm inner diameter and 100-mm length. Trimethylaminopropyl-bonded silica gel (BONDESIL-SAX, 40 μm, Varian, Palo Alto, CA) 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 Buffer C to Buffer D from 6 to 25 min and with Buffer D from 26 to 32 min. The flow rate was 2 ml/min, and the eluate was collected into eight 8-ml fractions. The fractions were concentrated to 0.5 ml by partial lyophilization (EYELA FD-81, Tokyo Rikakikai, Tokyo, Japan). The second dimensional chromatography was performed with a gel permeation chromatography (GPC) column (Bioassist G2SWXL, TOSOH, Tokyo, Japan). 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 (12 fractions). In this way a total of 96 fractions (8 x 12) were obtained.

Enzymatic Digestion for MD-LC-MS/MS - Samples in each fraction were reduced by incubating in 5 mM DTT at 60 °C 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 °C for 1 h. 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.

In-column Enzymatic Digestion - Proteins retained on the SAX column were treated with 0.1% RapiGest (Waters, Milford, MA) in 5 mM DTT and incubated at 60 °C for 30 min. The proteins were alkylated and digested with modified trypsin at 37 °C 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.