Human Heart (Ventricle) Database Human Heart (Ventricle) DatabaseMethods for the construction of the Harefield Human Heart (Ventricle) 2-DE Protein Database
1 Sample preparation
Samples of human ventricular myocardium were taken from explanted hearts at the time of cardiac transplantation and frozen in liquid nitrogen. The tissue specimens were crushed while still frozen between two cooled metal blocks. The resulting powder was homogenised in 1% w/v SDS in Eppendorf tubes, spun at 10,000 g for 5 min, rehomogenized and spun once more before harvesting the supernatant. The protein concentration of the sample was determined by the Bradford dye-binding procedure [1] and the samples stored at -80°C.
2 Analytical 2-DE
Analytical 2-DE was used to produce 2-D gels for silver staining or Western immunoblotting. This was performed using the ISO-DALT system [2, 3] as described in [4]. First-dimension IEF was carried out in 210 x 1.5 mm rod gels containing 3.2%T, 2.6%C polyacrylamide, 9M urea, 4% w/v CHAPS, and 2% w/v Resolyte pH 4-8 (Merck). Protein samples were mixed with at least twice their volume of lysis buffer (9M urea, 4% w/v CHAPS (Calbiochem), 1% w/v DTT, 2% w/v Resolyte pH 4-8 and 0.5% w/v Bromophenol blue) prior to application to the cathodic end of the IEF gels. For silver staining an 80 µg protein loading was used, while for Western blotting a loading of 200 µg was used. Gels were focused at 800 V for 35,000 Vh. After IEF, the gel rods were extruded onto strips of Parafilm and stored at -80°C. The second-dimension electrophoresis was performed on 230 x 200 x 1.5 mm 12%T SDS-PAGE gels without stacking gels using the standard Laemmli buffer system [23]. First-dimension rod gels were loaded on to the second-dimension gels slabs while still frozen and equilibrated in situ for 5 min with 1% w/v DTT, 2% w/v SDS, 0.05% Bromophenol blue, 0.125 M Tris base, pH 6.8. The second-dimension electrophoresis was carried out at 15 mA/gel overnight at 10°C.
3 Preparative 2-DE
Preparative 2-DE was used to produce 2-D gels for protein microsequencing. This was performed using the Investigator 2-DE system (Oxford Glycosystems) as described in [5]. First-dimension IEF was carried out in 210 x 3 mm rod gels containing 3.2%T, 2.6%C polyacrylamide, 9M urea, 4% w/v CHAPS, and 2% w/v Resolyte pH 4-8. Protein samples were mixed with at least twice their volume of lysis buffer (9M urea, 4% w/v CHAPS, 1% w/v DTT, 2% w/v Resolyte pH 4-8 and 0.5% w/v Bromophenol blue) prior to application to the cathodic end of the IEF gels. A protein load of 750 µg was used and gels were focused at 800 V for 35,000 Vh. After IEF, the gel rods were extruded onto strips of Parafilm and stored at -80°C. The second-dimension electrophoresis was performed on 230 x 200 x 1.5 mm 12%T SDS-PAGE gels with 2 cm 6%T stacking gels. First-dimension rod gels were equilibrated for 5 min with 1% w/v DTT, 2% w/v SDS, 0.05% Bromophenol blue, 0.125 M Tris base, pH 6.8. They were then loaded onto the second-dimension gels slabs using alignment guides [5]. The second-dimension electrophoresis was carried out at 3000 mW/gel overnight at 10°C.
4 Silver staining
After electrophoresis, analytical 2-D gels were fixed overnight in a methanol:acetic acid:water solution (5:1:4[v/v/v]). 2-D protein profiles were visualised by silver staining with the Daiichi 2-D Silver Staining Kit. Permanent records of silver-stained 2-D patterns were made by contact-printing onto X-ray duplicating film.
5 Western immunoblotting
Analytical 2-D gels were electroblotted onto nitrocellulose membranes (Hybond C Super, Amersham) at 500 mA for 6 h in 20 mM Tris base, 150 mM glycine buffer, pH 8.3. After electroblotting, 2-D blots were stained for 5 min with Instaview Nitrocellulose (Merck) to visualise the total protein pattern. A permanent record of the protein profile for identification of the protein spots visualised by subsequent immunoprobing was made by computerised densitometry. The blots were then destained using the Instaview destaining reagent. This stage completely destains the protein spots and the background, allowing subsequent immunoprobing of the blot.
Non-specific protein-binding sites on the nitrocellulose membranes were blocked with 3% w/v nonfat dried milk in PBS, containing 0.05% w/v Tween-20 (PBS-Tween) for 1 h. After washing in PBS-Tween, membranes were incubated with the primary antibody and agitated for 1 h. After thorough washing in PBS-Tween, the membranes were incubated for 1 h with the appropriate species-specific secondary antibody conjugated with horseradish peroxidase. Membranes were then washed thoroughly in PBS and protein spots reactive with the primary antibodies were visualised using an enhanced chemiluminescence (ECL) detection system [6] (Amersham International).
6 N-terminal microsequencing
After electrophoresis, preparative 2-D gels were equilibrated for 30 min in 50 mM Tris base, 50 mM boric acid buffer, pH 8.5 [7]. The gels were then electroblotted onto FluoroTrans membranes (Pall, Havant, UK) at 500 mA for 6 h at 10°C using the blotting accessories for the DALT tank [4]. The 2-D protein profiles were visualised by staining the blots with Coomassie Brilliant Blue (CBB) R-250. After destaining in 45% v/v methanol, 10% v/v acetic acid, 45% v/v water, the blots were rinsed in deionised water, air dried and sealed into plastic bags until used for N-terminal microsequence analysis.
The protein of interest were excised from the stained FluoroTrans membranes, rinsed with deionised water and placed into the Blott Cartridge [8] fitted to an ABI Model 477A pulsed-liquid protein sequencer (Applied Biosystems) equipped with an ABI Model 120A PTH analyser. Each protein was sequenced using an optimised FastBlot (FBlot-1) Programme (Applied Biosystems). The N-terminal protein sequences were compared for homologies to known proteins using the BLAST programme [9] in conjunction with the OWL composite protein database [10] at the Daresbury Laboratory SEQNET Facility (SERC, UK).
7 Internal microsequencing
Preparative 2-D gels for internal microsequence analysis were fixed overnight in a methanol:acetic acid:water solution (5:1:4 [v/v/v]). They were then stained with 0.1% w/v CBB G-250 in 2% w/v phosphoric acid, 10% w/v ammonium sulphate, and 20% v/v methanol [11]. This method uses CBB in a colloidal form so that no destaining step is required. The gels were stained until optimal sensitivity was obtained (usually about 16 h). Gels were stored in sealed polythene bags at 4°C until required. For internal digestion identified protein spots were cut from the gel with a cork borer or scalpel and placed in microcentrifuge tubes before destaining with 60% v/v CH3CN, 0.1% v/v TFA for 1 h at 37°C. The destaining solution was then aspirated and the gel reswollen in 200 µl NH4CO3 (200 mM, pH 8.9) for 30 min at 37°C. In gel digestion was performed essentially after the method of Rosenfeld et al. [12]. In brief, gel discs were placed in 200 µl of 200 mM ammonium carbonate solution in 60% v/v CH3CN, 40% H2O pH 6.9 for 30 min at 37°C and then partially air dried on foil for five to ten minutes. The gel slices were then swollen with 5 µl of ammonium carbonate buffer (200 mM, pH 6.9) followed by application of 5 µl of ammonium carbonate buffer containing 0.5 µg of sequencing grade modified trypsin (Promega). The gel slices were then placed in microcentrifuge tubes and two 5 µl aliquots of digestion buffer were added to further rehydrate the gels. After incubation at 37°C overnight the gels were extracted twice with 200 µl aliquots of 60% v/v CH3CN, 40% v/v H2O with 0.1% v/v TFA. The pooled extraction aliquots were dried in a centrifugal evaporator at 40°C and the dried digests stored at 4°C until required for HPLC analysis.
Dried digests were dissolved in 100 µl of 5% v/v CH3CN, 95% v/v H2O with 0.1% v/v TFA and fractionated by HPLC on an Aquapore RP300 reversed-phase column (Applied Biosystems) using an Applied Biosystems 130A separation system. Peptides were eluted using a linear gradient from 0 to 60% B over 40 min (solvent A = 5% v/v CH3CN, 95% v/v H2O with 0.1% v/v TFA; solvent B = 60% v/v CH3CN, 40% v/v H2O, with 0.08% v/v TFA) at a flow rate of 200 µl/min. Elution was monitored at 214 nm and 1 min fractions collected using a Jaytee fraction collector. For sequencing, peptide peaks were dried by centrifugal evaporation before dissolving the peptide in 100% TFA and application directly to quaternary amine derivatized PVDF discs (see below). After air-drying, discs were then sequenced as for blotted samples and sequence homologies determined as above. PVDF membranes were derivatized with quaternary ammonium groups by submerging 6 mm discs of FluoroTrans (Pall) under 5 ml 3-aminopropyl-trimethoxysilane (Aldrich) for two days at room temperature (method after that of P. Paroutaud, Applied Biosystems, France, personal communication). The membranes were then removed and washed in methanol (Rathburn, HPLC grade) four times, dried then placed in a vial containing iodomethane (Aldrich) for 2 h at room temperature. The membranes were then washed four times in methanol, dried and stored at room temperature until used.
8 Peptide Mass Profiling
For enzyme digestion, stained protein spots were excised from blots and destained by washing twice in 1ml 70% acetonitrile. Spots were then air dried and cut into approximately 1-2 mm squares and the fragments placed in clean microcentrifuge tubes. Enzymes were prepared in 25 mM ammonium carbonate, pH 7.8 containing 10 % methanol and 1 % octyl-B-glucoside (Oxford Glycosystems) to increase wetting of the membrane and to prevent adsorbtion of the enzyme to the membrane. Enzymes were dissolved at a concentration of 1 µg/µl and stored frozen at -20°C in aliquots until required for use when they were diluted to a concentration of 0.1 µg/µl in digestion buffer. The enzymes used were modified (methylated) trypsin and Lys-C which cleave at the C-terminal side of lysine and arginine, and lysine respectively. All enzymes were of sequencing grade (Promega).
One to four microlitres of enzyme working solution (sufficient to just wet the membrane) were used per protein spot [13]. Protein spots were digested overnight at 28°C followed by extraction twice in 50 µl of formic acid/ethanol (50:50 v/v) for 1 h. The pooled extracts were then dried by centrifugal evaporation at 40°C and stored at 4°C until used.
Samples for mass-spectrometry were dissolved in 5-20 µl of 10% methanol. An aliquot (2 µl) was then removed and one microlitre of alpha-cyano-4-hydroxy cinnamic acid (Aldrich; stock solution of 10 mg/ml in 70% acetonitrile) added. The internal standards Substance P ([M+H]+ = 1348.7) and oxidized insulin B-chain ([M+H]+ = 3496.7 Da) obtained from Sigma were dissolved in methanol and stored at 4°C until required. Prior to mass-spectrometry, an aliquot (1 µl) containing 200 fmol of each standard was added to each sample as internal calibrant. Samples (1 µl) were pipetted onto 48-position stainless steel sample strips and air dried before loading into the mass spectrometer. Spectra were obtained using a Lasermat 2000 laser desorption mass spectrometer (Finnigan MAT) just above the threshold for desorption/ionisation, and averaged for 10-30 laser pulses. Mass peaks were calibrated using manual internal standardisation and peptide masses used to search relevant peptide databases (created using MassMap, Finnigan MAT software) generated from the NCBI Entrez sequence database using the MOWSE algorithm [14] within MassMap. Searching was performed using a mass uncertainty of ±2-4 Da and a molecular weight range of ±15% of the Mr determined from 2-DE. It was not possible to filter the output for species or pI for searches performed with MassMap or MOWSE. The output consisted of a list of proteins ranked by a statistical score [14] and results for dual digests for each protein were combined using the Combine Searches utility within MassMap.
9 Amino acid compositional analysis
Amino acid analysis of PVDF-bound proteins was as described in [15,16]. Single PVDF spots were hydrolysed (6 M HCl at 155°C for 1 h), amino acids extracted, and the amino acid composition determined using Fmoc precolumn derivatisation on a GBC Automated Aminomate System [17]. Chromatography was according to Ou et al [18]. Percentage amino acid composition was determined for 16 amino acids (Asx, Glx, Ser, His, Gly, Thr, Ala, Pro, Tyr, Arg, Val, Met, Ile, Leu, Phe, Lys) by comparing pmole yield of each amino acid to the total pmol yield of all 16 amino acids.
Amino acid composition and estimated pI and Mr from 2-DE of each protein were matched against all human entries of the SWISS-PROT database by the program AACompID, using windows of pI ± 0.5 units and Mr ± 20%. Molecular weight estimations were modified in some cases to account for N-terminal truncation of molecules. AAcompID was accessed through the SWISS-2D PAGE menu of the ExPASy server, via the World Wide Web (http//:expasy.hcuge.ch/ch2d/aacompi.html) [19, 20]. Analyses were calibrated using a human serum albumin sample that had been hydrolysed, extracted and analysed at the same time as other spots.
10 Computerised densitometry
Silver stained 2-D gels and Instaview stained Western blot transfers were digitised at 176 µm resolution using a Molecular Dynamics 300A laser densitometer. Protein patterns were analysed and the 2-D gel protein database established using the PDQuest software package (Protein and DNA Imageware Inc) [21, 22] running on a Sun IPX workstation. Protein database SSP (sample spot protein) numbers were assigned to unique protein spots by PDQuest. Hard copies of computer images of the annotated database were generated using IslandPaint and IslandDraw running on a Sun IPX workstation.
2-D gels were calibrated for Mr and pI using a group of known proteins appearing on the standard heart pattern. The Mr was obtained from the primary amino acid sequence using the Daresbury SEQNET facility, and pI was calculated from this sequence using the "Isoelectric" programme in the GCG package. Mr and pI values for the remaining spots were calculated automatically by PDQuest.
SSP Protein Name Mr pI 105 Troponin C 18,500 3.6 1104 Myosin light chain 2 21,000 5.0 2108 Myosin light chain 1 26,000 5.2 317 Tropomyosin 42,200 4.4 2410 Actin 34,000 5.4 5726 Albumin 66,000 6.0 6810 Vinculin 116,000 6.4 8424 Creatine kinase M 41,900 7.0 8013 Haemoglobin 14,700 7.3 6002 Fatty acid binding protein 15,000 6.3References