|
Chemicals |
Company |
Ordering number |
|
RPMI 1640 (1*with L-glutamine) |
Biorad |
F1215 |
|
fetal calf serum |
Gibco/BRL |
10270-106 |
|
streptomycin |
Grünenthal |
757753B |
|
penicillin |
Grünenthal |
E744114 |
|
cyclosporine A, SandimmunR |
Sandoz |
PZN-2702663 |
|
LiqueminR 25 000 U/ml |
Hoffmann-La Roche |
PZN-3441331 |
|
FicollR separating solution (density 1.077 g/ml) |
Seromed |
L6115 |
|
dimethylsulfoxide (DMSO) |
Merck |
9678.0100 |
|
colcemid |
Gibco/BRL |
15210-016 |
|
culture medium |
RPMI 1640 |
500 ml |
|
fetal calf serum |
50 ml | |
|
streptomycin |
50 mg | |
|
penicillin |
50,000 U | |
|
transformation medium |
filtered EBV-containing B95-8 supernatant |
50 ml |
|
RPMI 1640 |
40 ml | |
|
fetal calf serum |
10 ml | |
|
streptomycin |
50 mg | |
|
penicillin |
50,000 U | |
|
cyclosporin A (SandimmunR) |
100 µg | |
|
hypotonic solution |
KCl |
0.075 M |
|
freezing medium |
culture medium |
9 ml |
|
DMSO |
1 ml | |
|
cold fixative |
methanol |
3 vol |
|
acetic acid |
1 vol |
|
Equipment |
Company |
|
tissue culture flasks (50 ml) |
Nunc |
|
sterile filters Millex HA |
Millipore |
|
centrifuge with 3360/BS4402/A rotor |
Heraeus sepatech |
The protocol has been established by Neitzel (1986). Fig. 3-1 depicts the procedure. The Epstein-Barr-Virus (EBV) for transformation is obtained from the lymphoblastoid marmoset [page 20↓](monkey) cell line B95-8 that is latently infected with EBV and can release virus particles into the culture medium. About 5 x 105 B95-8 cells/ml were suspended in culture medium and the EBV-containing supernatant medium was collected after 5 days of cultivation. Then the supernatant was centrifuged at 400 g for 10 min to remove marmoset cells completely. After additionally being filtered twice through a 0.45 µm membrane filter, the supernatant was diluted 1:1 with fresh RPMI 1640, supplemented with 20% heat-inactivated fetal calf serum, 2 mM L-glutamine, antibiotics and cyclosporin A, to obtain the virus pools, i.e. the transformation medium.
The heparinated blood sample (500 IE Liquemin/ml blood) of a normal control patient was diluted 1:1 with RPMI 1640 and was overlaid on 5 ml Ficoll. The volume ratio of blood-
| Fig. 3-1: Establishment of lymphoblastoid cell lines . The left column depicts the preparation of the transformation medium. The right column depicts the preparation of the mononuclear cells. The final bottle contains both the transformation medium and the mononuclear cells, which are set up for cultivation of a lymphoblastoid cell line. | ||
|
|
RPMI mixture to Ficoll is 3-4:1. The step-gradient was centrifuged without brake at 400 g for 40 min in a swinging bucket centrifuge. The ensuing ring of mononuclear cells at the interface [page 21↓] between plasma and Ficoll was aspirated and washed/centrifuged (400 g, 10 min) three times with 10 ml RPMI.
The mononuclear leukocytes, including T- and B-lymphocytes were suspended in transformation medium. The pH of the cell culture was adjusted to 6.8 by overlaying it with CO2 and then the cells were incubated at 37°C in a 5% CO2 atmosphere. The medium was changed once a week by replacing half of the supernatant with a fresh medium containing 1 µg/ml cyclosporin A. Cyclosporin A specifically inhibits the RNA polymerase of T lymphocytes thus removing the interfering T-lymphocytes.
The lymphoblastoid cell pellet was prepared after the cell line had been cultured for 3-4 weeks. The total cell number was counted with a Neubauer’s chamber under a phase contrast microscope at 100 x magnification. The cultured cells were re-suspended by pipetting up and down and were transferred into centrifugation tubes. A pellet was obtained by centrifugation at 1050 g for 8 min.
|
Chemicals |
Company |
Ordering number |
|
sucrose |
Merck |
7653.1000 |
|
EDTA |
Merck |
8418.0250 |
|
3-morpholinopropanesulfonic acid (MOPS) |
Fluka |
69948 |
|
bovine serum albumin (BSA) |
Fluka |
05488 |
|
triethanolamine |
Fluka |
90278 |
|
digitonin |
Fluka |
37006 |
|
Percoll |
Fluka |
77237 |
|
Metrizamide |
Fluka |
69753 |
|
medium A |
sucrose |
100 mM |
|
EDTA |
1 mM | |
|
MOPS (pH 7.4) |
20 mM | |
|
BSA |
1 g/l | |
|
medium B (homogenization buffer) |
medium A |
|
|
triethanolamine |
10 mM | |
|
Percoll |
5% | |
|
digitonin |
0.1 mg/ml |
The present protocol has been adapted and modified from Madden et al.(1987), Bourgeron et al.(1992) and Strack et al. (2001). Fig. 3-2 shows the procedure and the result of mitochondrial isolation.
| Fig. 3-2 : Mitochondrial isolation by Percoll/metrizamide hybrid gradient centrifugation. Electron microscopy reveals that the first fraction contains mostly membrane debris from both cellular and subcellular organelles. The material of the second fraction contains a few lysosomes and other structures that are difficult to identify morphologically. Only the third fraction contains highly enriched mitochondria. | ||
|
|
All procedures were carried out at 4°C to minimize protease activity. At first the cell pellet was washed and centrifuged (1050 g, 8 min) two times with medium A. Then it was resuspended with medium B and incubated for 5 minutes. Medium B contains digitonin that is incorporated into the cell membrane and thus makes the subsequent break-up of cells easier. The cells were disrupted with 5 up- and down-strokes of a Potter-Elevhjem-homogenizer at 500 rpm. The homogenate was centrifuged at 1300 g for 5 min, and the supernatant was collected. The pellet was resuspended again in medium B and disrupted once more with the homogenizer. This step was repeated twice and the supernatants from each centrifugation-step were pooled as the “post-nuclear supernatant”.
A hybrid gradient was prepared from 35% Metrizamide (density 1.1304 g/ml), 17% Metrizamide (density 1.1029 g/ml), and 6% Percoll (density 1,0331 g/ml). All solutions were pre[page 23↓]pared with 0.25 M sucrose and the concentrations were expressed as w/v. Gradients were poured into cellulose-nitrate ultracentrifugation-tubes. 1 ml of 35% Metrizamide was overlaid with 1 ml of 17% Metrizamide followed by 2.5 ml of 6% Percoll. The tubes were then gently filled with post-nuclear supernatant (about 3.7 ml) up to 4 mm below the upper rim. The solutions were overlaid on top of one another with a long lumbar puncture syringe-needle. Centrifugation was performed at 20,000 g without brake for 15 min at 4°C in an ultracentrifuge.
After centrifugation several distinct bands can be detected in the discontinuous hybrid Percoll/Metrizamide gradient. The band at the interface between 17% and 35% Metrizamide is highly enriched in mitochondria [Madden et al., 1987; Strack et al., 2001]. This band was aspirated with a Pasteur-pipette, diluted 1:4 with 0.25 M sucrose and centrifuged at 10,000 g for 10 min to wash away the remaining Metrizamide and to obtain a concentrated mitochondrial pellet at the floor of the centrifugation tube. An additional washing step could be added if necessary.
|
Chemicals |
Company |
Ordering number |
|
NaH2PO4*2H2O |
Merck |
6580 |
|
Na2HPO4 |
Merck |
3090 |
|
KCl |
Merck |
4933 |
|
MgSO4*7H2O |
Sigma |
M-1880 |
|
urea |
Biorad |
161-0730 |
|
pepstatin A |
Sigma |
P-4265 |
|
phenylmethylsulfonylfluoride |
Biorad |
161-0202 |
|
protease inhibitor mini (CompleteR) |
1.836.153 | |
|
glycerin |
Merck |
4093 |
|
3-[(3-cholamidopropyl)-dimethylammonio] -propan-sulfonate (CHAPS) |
Merck |
1.11662.0001 |
|
1,4-dithioerythritol (DTT) |
Biorad |
161-0610 |
|
ServalyteR pH 2-4 |
Serva |
42902 |
|
Equipment |
Company |
|
sonicator (Transsonic 310) |
Faust |
|
polished glass beads (diameter 2.5 mm) |
Carl Roth GmbH + Co Karlsruhe |
The protocol was performed according to Klose et al. (1999a). The mitochondrial pellet was weighed to calculate the required volume of each solution, including P2-CHAPS/MgSO4, protease inhibitor 1 (H1), protease inhibitor 4 (H4), and the required number of glass beads. The required volume of P2-CHAPS/MgSO4 was 1.25 times the weight of the mitochondrial pellet.
H1 protease inhibitor was calculated to be 0.02 fold and H4 protease inhibitor to be 0.08 fold of the combined masses of the mitochondrial pellet and P2-CHAPS/MgSO4. The number of the required glass-beads equaled to 0.034 x of the sum of the weight of mitochondrial pellet plus the volumes of all other solution components. The solutions were added directly onto the pellet. After adding the glass-beads to the homogenate, it was sonicated in an ice-cold water bath for 10 seconds followed by 40-45 seconds stirring and one minute keeping on ice. This sonication-round was repeated six times to guarantee that most of the mitochondrial proteins were solubilized. After 15 min stirring in a cold room, the required volume of benzonase was added to remove the mtDNA. The volume of the required benzonase equaled 0.025 x the weight of the homogenate after sonication. The homogenate was stirred for another 15 min at 4°C before the protein-concentration was measured by the BCA protein-assay. This assay required about 1-5 µl homogenate. The rest of the homogenate was mixed with urea, DTT and ampholine pH 2.0-4.0 to yield final concentrations of 9 M urea, 70 mM DTT, and 2% ampholine 2.0-4.0. The homogenate was stored at –80°C before the 2D-electrophoresis was started.
|
Chemical |
Company |
Ordering number |
|
BCA protein assay reagent (includes reagent A, reagent B, and albumin standard) |
Pierce |
23225BN |
|
Equipment |
Company |
|
spectrophotometer (MRX) |
Dynal Biotech & Nordic |
|
microwell-plate |
NUNC TM Brand Products |
|
|
[page 25↓] |
The BSA standards were prepared by diluting a 2.0 mg/ml BSA stock standard serially with the same diluent as my sample. A list of standard dilutions with a working range from 20 µg/ml to 2000 µg/ml is shown bellow:
|
Volume of BSA |
Volume of diluent |
Final BSA concentration |
|
300 µl of Albumin Standard |
0 µl |
2000 µg /ml |
|
(A) 375 µl of albumin standard |
125 µl |
1500 µg /ml (A) |
|
(B) 325 µl of albumin standard |
325 µl |
1000 µg /ml (B) |
|
(C) 175 µl of (A) |
175 µl |
750 µg /ml (C) |
|
(D) 325 µl of (B) |
325 µl |
500 µg /ml (D) |
|
(E) 325 µl of (D) |
325 µl |
250 µg /ml (E) |
|
(F) 325 µl of (E) |
325 µl |
125 µg /ml (F) |
|
(G) 100 µl of (F) |
400 µl |
25 µg /ml (G) |
The BCA working reagent was prepared by mixing 50 parts of BCA protein assay reagent A (contains BCA) with one part of reagent B (contains CuSO4). Then 20 µl of each standard, the sample or diluent (as empty control) were pipetted into wells of a microwell-plate. 400 µl working reagent were added into each well sequentially. The plate was then covered and incubated at 37°C for 30 minutes in a water bath. After incubation, the plate was cooled to room temperature before final measurement. The protein concentration was measured colorimetrically at λ=570 nm with a spectrophotometer. The program “Revelation Version 2.0” provided by the manufacturer was used for data processing.
|
ampholine-mix |
Pharmalyte pH 3.5-10 |
12.5% (v/v) |
|
Sevalyte pH 2.0-11 |
12.5% (v/v) | |
|
Pharmalyte pH 6.5-9.0 |
12.5% (v/v) | |
|
Pharmalyte pH 4.0-6.5 |
37.5% (v/v) | |
|
Pharmalyte pH 5.0-8.0 |
25% (v/v) | |
|
1D-separation gel |
acrylamide |
3.5% (w/v) |
|
piperazine diacrylamide |
0.3% (w/v) | |
|
urea |
9 M | |
|
TEMED |
0.06% (v/v) | |
|
glycerin |
5% (w/v) | |
|
ampholine-mix |
4% (v/v) | |
|
persulfate |
0.02% (w/v) | |
|
1D-anode solution |
urea |
3 M |
|
phosphoric acid |
0.742 M | |
|
1D-cathode solution |
urea |
9 M |
|
glycerin |
5% (w/v) | |
|
ethylenediamine |
0.749 M | |
|
Sephadex mixture |
Sephadex G-200 |
12.5% (w/v) |
|
glycerin |
12.5% (w/v) | |
|
DTT |
1.08% (w/v) | |
|
ampholine-mix |
2% (v/v) | |
|
urea |
9 M | |
|
1D-incubation solution |
Tris-base |
125 mM |
|
g lycerin |
40% (w/v) | |
|
DTT |
65 mM | |
|
SDS |
3% (w/v) | |
|
2D-gel solution |
acrylamide |
15% (w/v) |
|
bisacrylamide |
0.2% (w/v) | |
|
Tris-base/Tris-HCl |
0.375 M | |
|
TEMED |
0.03% (v/v) | |
|
SDS |
0.1% (w/v) | |
|
persulfate |
0.08% (w/v) | |
|
2D-electrode solution (both upper and lower) |
Tris-base |
0.025 M |
|
glycine |
0.192 M | |
|
SDS |
0.1 M | |
|
2D-fixation solution (silver stain) |
ethanol |
50% (v/v) |
|
acetic acid |
10% (v/v) | |
|
2D-fixation solution (Coomassie stain) |
methanol |
50% (v/v) |
|
phosphoric acid |
2% (v/v) |
|
|
[page 27↓] |
|
Equipment |
Company |
|
apparatus for IEF and 40 cm glass tubes of two different diameters (0.9 mm and 1.5 mm) Fig. 3-3A |
self built |
|
apparatus for SDS-PAGE (Desaphor VA 300) Fig. 3-3B |
DESAGA |
|
circular cooling machine F25 |
Julabo |
|
PowerPac 3000 electrophoresis power supply |
Biorad |
|
1D-gel tube stand with special gel-solution groove |
self built |
|
polymerization stand Desaphor VA (2D polymerization stand) |
DESAGA |
|
1D-precision glass tubes 40 cm x 0.9 mm or 1.5 mm |
Schott |
|
pH-meter 766 with a micro electrode (type Inlab 422) |
pH-meter: Knick electrode: Roth |
| Fig.3-3A : equipment for isoelectric focusing | ||
|
|
| Fig. 3-3B : equipment for SDS-PAGE | ||
|
|
The isoelectric focussing is performed according to a protocol by Klose (1999b). In the first dimension two alternative gel containers were used. These are high precision capillary glass tubes with an internal diameter of 0.9 mm (thin) or 1.5 mm (thick) and a length of 400 mm. Gel solution was filled into the tubes by using accurately fitting nylon strings as plungers. After filling, the tubes were kept at room temperature to polymerize for 3-4 days before use. Before the protein samples were loaded onto the anodic end of the IEF-gel, a Sephadex mixture – acting as a sieve – was loaded to a height of 2 mm. Gels were electrophoresed serially at 100 V for 1 hour, followed by 300 V for 1 hour, 1000 V for 23 hour, 1500 V for 30 min and finally at 2000 V for 10 min. After the IEF-run was finished, the gels were expelled directly into the 1D-incubation solution by a nylon string. They were incubated for 10 min at room temperature under continuous shaking and then placed completely relaxed onto the gel [page 28↓]grooves and stored at –70°C until the second dimension separation of the 2D-electrophoresis was performed.
In the second dimension, 0.75 mm (thin) or 1.5 mm (thick) thick plastic spacers were used to fix the distance between the two glass plates of the electrophoretic cell. The first dimension (IEF) gel was gently transferred from the groove onto the surface of the SDS-PAGE gel with the help of a special wire-hook. Care was taken, not to stretch the gel. The IEF gel had to be in tight contact with the SDS-PAGE gel. The inclusion of air or solution between the gels had to be avoided. 1 % agarose solution was overlaid up to the edges of the glass cells to restrict the movement of the IEF gel. Gels were electrophoresed for the first 15 min at 65 mA (thin gel) or 130 mA (thick gel) and then at 75 mA (thin gel) or 150 mA (thick gel) for ca. 6-7 hours. The temperature of the lower electrode solution was kept at 15°C by a spiral glass tube fixed to a circular cooling pump. Electrophoresis was finished when the bromophenol blue line in the gels reached a line that has been etched 2 cm from the lower edge of the frontal gel plate. After electrophoresis, the gels were transferred into 1 liter/gel 2D-fixation solution. After shaking for 2 hours, the gels were left standing overnight in the same solution at room temperature.
The isoelectric focussing of two Ø 1.5 mm tube gels was performed as described above. One of the gels was loaded with 10 µl of mitochondrial protein sample, the other was left empty as a control. After the isoelectric focussing was finished, the gels were expelled and cut into 5 mm sections which were put directly into individual Eppendorf test tubes with 40 µl degassed aqua bidest. The Eppendorf tubes were closed in a nitrogen atmosphere. The gel sections were sonicated in an ice-cold water bath for 15 min in order to release the ampholytes from the gel into the water. The pH-measurement was carried out with a microelectrode. The pH of each gel-section was measured for 2 min until stable readings were obtained.
|
Chemicals |
Company |
Ordering number |
|
sodium acetate |
ICN |
195496 |
|
sodium thiosulfate |
ICN |
191447 |
|
glutardialdehyde |
Merck |
8.20603 |
|
ethanol |
Herbeta |
21847 |
|
sodium carbonate |
Merck |
1.06392.0500 |
|
silver nitrate |
ICN |
195495 |
|
formaldehyde |
ICN |
194047 |
|
EDTA |
Merck |
1.08418.0250 |
|
thimerosal |
ICN |
103044 |
|
ammonium sulfate |
Sigma |
A-9141 |
|
Serva Blue G-250 R |
Serva |
35050 |
|
|
[page 29↓] |
|
S-incubation solution |
sodium acetate |
0.5 M |
|
sodium thiosulfate |
0.2% (w/v) | |
|
glutardialdehyde |
0.5% (v/v) | |
|
ethanol |
30% (v/v) | |
|
S-stain solution |
silver nitrate |
0.1% (w/v) |
|
formaldehyde |
0,01% (v/v) | |
|
S-wash solution |
sodium carbonate |
2.5% (w/v) |
|
S-developer solution |
sodium carbonate |
2.5% (w/v) |
|
formaldehyde |
0.01% (v/v) | |
|
S-stop solution |
EDTA |
0.05 M |
|
thimerosal |
0.02% (w/v) | |
|
C-incubation solution |
methanol |
34% (v/v) |
|
phosphoric acid |
2% (v/v) | |
|
ammonium sulfate |
17% (w/v) | |
|
C-stain solution |
methanol |
34% (v/v) |
|
phosphoric acid |
2% (v/v) | |
|
ammonium sulfate |
17% (w/v) | |
|
Serva Blue G-250 |
0.066% (w/v) | |
|
C-wash solution |
methanol |
25% (v/v) |
|
Equipment |
Company |
|
shaker (3016) |
Gesellschaft für Labor mbH |
|
plastic troughs (bottom 30*40 cm) |
Brukle-Labo-Plast |
|
drying/vacuum incubator (type UL-60) |
Memmert |
|
water-saving vacuum pump (type TOM JET 1/A4) |
Genser Wissenschaftliche Apparate |
|
water-jet vacuum pump |
Th. Geyer |
|
cellophane |
Gehring & Neidweiser GmbH & Co |
The silver staining was performed according to a protocol by Klose (1999b). Silver staining is a very sensitive method with a detection limit between 1-10 ng. It is based on the high reducibility of silver ions. The silver ions form complexes with proteins much stronger than with the polyacrylamide gel. Complexed silver ions can be reduced much faster than free silver ions. During the whole procedure of silver staining, the gels were shaken continuously. After each step, the solutions were removed by suction of a water-jet pump. The gels were at first incubated in S-incubation solution for 2 hours. During this period, the sodium thiosulfate and glutardialdehyde in the solution act as complexing agents and link the proteins by forming covalent bonds. After that, two rinsing steps with distilled water were performed for 20 min. This had to be done to minimize background staining by washing away the unbound glutaraldehyde. The silver staining lasted for 30 min. In this step formaldehyde was used as reducing agent. After that, the gels were washed in S-wash solution for 1 min and developed by S-developer solution for several minutes and then finally stopped by S-stop solution.
|
|
[page 30↓] |
The colloidal Coomassie staining was performed according to a protocol by Klose (1999b). Coomassie staining is a method that visualizes proteins due to the unspecific binding of the dye to their amino acid residues. The detection limit is around 1 μg. Compared to the standard Coomassie staining method, the colloidal Coomassie staining is more specific and sensitive, since the colloidal Coomassie dye is much finer than the standard Coomassie dye. The colloidal Coomassie dye penetrates better through the polyacrylamide gel and thus binds to the proteins more easily. The whole procedure of colloidal Coomassie staining was carried out under continuous shaking. After overnight fixation in C-solution, the gels were at first washed three times with distilled water for 30 min. Then they were incubated in C-incubation solution for 1 hour followed by 5 days staining with Coomassie brilliant blue G-250. The destaining step with C-wash solution lasted 1-2 hours by using a piece of sponge which acts as adsorbent for the washed out dye. The whole procedure was stopped when the protein spots stood out clearly from the background. One should not destain the gels for too long since the color of the protein spots also faded with time.
After staining the results had to be stored and the gels had to be preserved. Still wet, the gels were scanned on a transilluminating scanner and stored as TIFF-files with a resolution of 150-300 dpi. Later the gels were dried to preserve them for the records. They were “sandwiched” between two sheets of wet cellophane and thick filter papers and were put on a drying panel. Excess water as well as air bubbles between the layers of filter paper, cellophane, and the gel were expelled with a ruler. The gels were then dried in a heated vacuum incubator for approximately 2-3 hours at 80°C. The dried gels were labeled with the sample name and the date and stored in large envelopes tagged with all the information of the 2D-electrophoresis runs.
|
Chemicals |
Company |
Ordering number |
|
trypsin |
1047841 | |
|
ammonium bicarbonate (NH4HCO3) |
Sigma |
A-6141 |
|
acetonitrile |
Baker |
9017-54 |
|
formic acid |
Sigma |
F-0507 |
|
α-cyano-4-hydroxy cinnamic acid |
Sigma |
C2020 |
|
trifluoroacetic acid (TFA) |
Merck |
8178.0050 |
|
Equipment |
Company |
|
skin-biopsy punch (various diameters) |
Stiefel |
|
vacuum centrifuge (Speedvac) (PLCT 60-E) |
Heraeus |
|
incubator (PersonalHyb) |
Stratagene |
|
shaker (AVM) |
ETS Jean Robin |
Protein spots were excised from the gel with a skin-biopsy punch and placed into the destaining solution. After shaking overnight at room temperature, they were dehydrated by addition of 100 µl acetonitrile. The liquid phase was removed, and the gel pieces were completely dried in a vacuum centrifuge. The gel pieces were then re-hydrated in the trypsin solution at 4°C for 45 min to let the trypsin permeate into the gel pieces without self-digestion. The digestion was allowed to proceed overnight at 37°C by keeping the gel pieces wet. Peptides were extracted by letting the gel-piece swell three times with 5% formic acid and shrink four times with acetonitrile. The whole liquid phase was collected and finally dried down in a vacuum centrifuge.
The peptide-samples were solved in 10 µl 0.1% TFA. 1 µl of the sample was spotted onto the MALDI target plate and mixed with 1 µl of 2% TFA and 1 µl matrix. After the sample spots had air-dried completely, they were rinsed twice with 5-10 µl 0.1% TFA, and the remaining liquid was evaporated with pressurised air.
MALDI-TOF mass spectrometer (Reflex II from Bruker-Daltonik, Bremen)
Mass spectra of the peptide mixture were obtained using the Bruker Reflex II mass spectrometer operated in the reflector mode. The instrument is fuctions in the “delayed extraction” mode that ensures a mass resolution up to at least 6000 Da over the entire mass range and a mass accuracy of better than 0.1 Da with internal calibration. A total of 100-140 single-shot spectra were accumulated from each sample. They were calibrated using the monoisotopic peak from a known auto-digestion product of bovine trypsin (residues 50-69, M+H+ = 2163.06 Da) and the matrix trimer ion (3M+H+ = 568.14 Da) as internal standards. The XMASS 5.0 software packages provided by the manufacturer were used for data processing.
|
|
[page 32↓] |
The identification of proteins by their peptide mass fingerprints was mainly performed with the Mascot Software (Matrix Science Ltd.) and additionally with ProFound or PeptideSearch as search engines. The parameters were choosen as shown in Fig. 3-4.
| Fig. 3-4A: the first step of database search for proteins . Example of the parameters used for the database search with the Mascot search engine. The parameters such as taxonomy, allowed missed cleavages, variable modifications, and peptide mass tolerance were restricted as shown. Only the monoisotopic peptide masses were considered in this search. | ||
|
|
| Fig. 3-4B: the second step of database search for proteins . The first protein is usually the best fit. The full name of the candidate protein together with its gi-number, the theoretical molecular weight, the probability based Mowse-score and the number of matched peptides are all listed. Normally, there are several peptides or proteins listed in one suggestion. They generally are various fragments of the same protein. | ||
|
|
|
|
[page 33↓] |
| Fig . 3-4C : The third step of the database search . The detailed view of a certain protein, e.g. the ATP synthase beta-chain, includes information on the protein and on the sequence of the protein covered by the peptide mass fingerprint (highlighted in red). The full information of the protein can be accessed from the NCBI database, by clicking on the accession number (gi number) of the protein. | ||
|
|
After a protein had been found, the fingerprint data were compared with the theoretical digestion product of the protein. If no clear relation of the molecular weights between the “experimental” and the “theoretical” fragments could be found, I tried to use several less stringent criteria to improve the matching rates. This was expecially the case for large peptide fragments with molecular weights above 2,000 Da. The less stringent criteria comprised the allowance of up to four missed cleavages, the modification of cysteine by acrylamide and a larger tolerance of mass deviation of ± 0.5 Da.
|
Chemicals |
Company |
Ordering number |
|
acetonitrile(HPLC-grade) |
Baker |
9017-54 |
|
2,5-dihydroxybenzoic acid |
Sigma |
G-5254 |
|
isopropanol |
Merck |
1.09634.1000 |
|
POROS 10 R2 reversed-phase chromatography medium R |
PerSeptive Biosystems |
1-1118-02 |
|
matrix |
2,5-dihydroxybenzoic acid |
5 mg/ml |
|
acetonitrile |
3 vol | |
|
0.1% (v/v) TFA |
7 vol | |
|
POROS 10 R2; reversed-phase chromatography medium solution |
POROS 10 R2 reversed-phase chromato-graphy medium saturated with isopropanol |
|
|
[page 34↓] |
|
Equipment |
Company |
|
GELoader pipette tip |
Eppendorf |
|
plastic syringe (1.25 ml) |
Eppendorf |
|
API QSTAR Pulsar I mass spectrometer equipped with a MALDI ion source |
Applied Biosystems/MDS Sciex |
Sample purification was performed according to a protocol by Gobom (2001). A long, narrow pipette tip packed with 0.3 ml POROS 10 R2 reversed-phase medium served as a chromatography column. All the sample liquid was driven through the column by a disposable plastic syringe. Prior to use, the column was washed with 15 µl of acetonitrile-0.1 % TFA (8:2 v/v) followed by an equilibration step with 10 µl of 0.1% (v/v) TFA. The peptide sample was acidified with 2 µl 2% (v/v) TFA to obtain a final concentration of about 0.2-0.5 % (v/v). Then the sample was loaded onto the column and was slowly pumped over the reversed-phase medium. A washing step was performed with 10 µl 0.1% TFA, and the column was emptied completely by pressing air through it for a few seconds. Finally, 3 µl matrix as an eluent were loaded on the column and the eluate was loaded directly onto the target of the MALDI-QTOF mass spectrometer.
These experiments were performed within the selection cell (Q1) and the collision cell (Q2). All ions were transmitted resulting in the measurement of the entire mass range. The ion of interest was selected at first in cell Q1, then this precursor ion was split in the collision cell Q2 using argon as a collision gas. The ensuing fragments were analyzed in the TOF section of the instrument. The instrument was calibrated externally with peptides of known masses. The data processing was done with the “ANALYST” software packages provided by the manufacturer.
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