[page 19↓]

3.  Materials and methods

3.1. Preparation of lymphoblastoid cell pellets

3.1.1. Chemicals and reagents

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

3.1.2. Solutions

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

3.1.3. Special equipment

Equipment

Company

tissue culture flasks (50 ml)

Nunc

sterile filters Millex HA

Millipore

centrifuge with 3360/BS4402/A rotor

Heraeus sepatech

3.1.4. Procedure

3.1.4.1. Preparation of transformation medium

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.

3.1.4.2. Preparation of mononuclear leukocytes from whole blood

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.

3.1.4.3. Establishment of the permanent cell culture

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.

3.1.4.4. Preparation of the lymphoblastoid cell pellet

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.

3.2. Preparation of mitochondria

3.2.1. Chemicals and reagents

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

3.2.2. Solutions

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

3.2.3. Special equipment

Equipment

Company

motor driven tightly fitting Potter-Elvehjem homogenizer

RZR 2051 (glass tube with Teflon pestle, diameter 6,8 mm)

Heidolph

lumbar puncture syringe-needles (length: 7 cm, volume: 1 ml)

Braun Melsungen AG

ultracentrifuge with T1270 rotor (Sorvall ultra Pro 80)

DuPont de Nemours

GmbH

3.2.4. Procedure

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.

3.2.4.1. Preparation of the post-nuclear supernatant

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”.

3.2.4.2. Preparation of a hybrid Percoll/Metrizamide discontinuous gradient

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.

3.2.4.3. Preparation of the mitochondrial pellet

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.

3.3.  Sample preparation of mitochondrial proteins

3.3.1. Chemicals and reagents

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)

Roche

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

3.3.2. Solutions

P2-CHAPS buffer

KCl

0.2 M

glycerin

20%

phosphate buffer (0.2 M NaH2PO4 and 0.2 M Na2HPO4, pH 7.2)

0.1 M

P2-CHAPS/MgSO4

P2-CHAPS buffer

 

MgSO4*7H2O

1 mM

CHAPS

0.14 M

protease inhibitor (H1)

(solved in ethanol)

pepstatin A

1.4 µM

phenylmethylsulfonylfluoride

1.0 mM

protease inhibitor 4 (H4)

P2-CHAPS-Buffer

0.4 ml

protease inhibitor mini (CompleteR)

1 tablet

DTT solution

DTT

21.6%

3.3.3. Special equipment

Equipment

Company

sonicator (Transsonic 310)

Faust

polished glass beads (diameter 2.5 mm)

Carl Roth GmbH + Co Karlsruhe

3.3.4. Procedure

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.

3.4. Bicinchoninic acid (BCA) protein assay

3.4.1. Chemicals and reagents

Chemical

Company

Ordering number

BCA protein assay reagent (includes reagent A,

reagent B, and albumin standard)

Pierce

23225BN

3.4.2. Special equipment

Equipment

Company

spectrophotometer (MRX)

Dynal Biotech & Nordic

microwell-plate

NUNC TM Brand Products


[page 25↓]

3.4.3.  Procedure

3.4.3.1. Preparation of diluted BSA serial standards

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)

3.4.3.2. Protein quantification assay

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.

3.5. Two-dimensional protein electrophoresis

3.5.1. Chemicals and reagents

Chemicals

Company

Ordering number

acrylamide

Biorad

161-0100

piperazine diacrylamide

Biorad

161-0202

TEMED

Biorad

161-0800

persulfate

Biorad

161-0700

urea (for gel solution)

Biorad

161-0730

urea (for electrode solutions)

Merck

8484

glycerin

Merck

4093

ethylenediamine

Merck

800947

85% phosphoric acid

Merck

573

Sephadex G-200R

Pharmacia

17-0081-01

Tris-base

Sigma

T-1503

Tris-HCl

Sigma

T-3253

sodium dodecyl sulfate (SDS)

Merck

1.06498

glycine

Serva

23390

PharmalyteR pH 3.5-10

Pharmacia

80-1125-87

PharmalyteR pH 6.5-9.0

Pharmacia

17-454-01

PharmalyteR pH 4.0-6.5

Pharmacia

17-452-01

PharmalyteR pH 5.0-8.0

Pharmacia

17-453-01

SevalyteR pH 2.0-11

Serva

42-900

AmberliteR IRN-150

Serva

1-0341

ethanol

Herbeta

21847

methanol

Baker

8045

acetic acid

Merck

8.00947

3.5.2. Solutions

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↓]

3.5.3. Special equipment

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

3.5.4. Procedure

3.5.4.1. First dimension-isoelectric focussing (IEF)

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.

3.5.4.2. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis

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.

3.5.4.3. Measurement of the pH-gradient of the IEF-gel

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.

3.6. Gel staining and drying

3.6.1. Chemicals and reagents

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↓]

3.6.2.  Solutions

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)

3.6.3. Special equipment and material

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

3.6.4. Procedure

3.6.4.1. Silver staining

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↓]

3.6.4.2.  Colloidal Coomassie staining

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.

3.6.4.3. Gel drying and preserving

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.

3.7. Sample preparation for MALDI-TOF protein mass fingerprinting

3.7.1. Chemicals and reagents

Chemicals

Company

Ordering number

trypsin

Roche

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

3.7.2. Solutions

destaining solution

100 mM NH4HCO3

60% (v/v)

acetonitrile

40% (v/v)

trypsin solution

trypsin

10 ng/µl

NH4HCO3

50 mM

formic acid solution

formic acid

5% (v/v)

matrix

α-cyano-4-hydroxy cinnamic acid

15 mg/ml

0.1% (v/v) TFA

30% (v/v)

acetonitrile

70 % (v/v)

3.7.3. Special equipment

Equipment

Company

skin-biopsy punch (various diameters)

Stiefel

vacuum centrifuge (Speedvac) (PLCT 60-E)

Heraeus

incubator (PersonalHyb)

Stratagene

shaker (AVM)

ETS Jean Robin

3.7.4. Procedure

3.7.4.1. In-gel digestion

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.

3.7.4.2. Sample preparation for MALDI analysis

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.

3.8. Peptide mass fingerprinting by MALDI-TOF mass spectrometry

3.8.1. Special equipment

MALDI-TOF mass spectrometer (Reflex II from Bruker-Daltonik, Bremen)

3.8.2. Procedure

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↓]

3.9. Computer aided analysis of protein mass fingerprints

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.

3.10. Peptide sequencing by MALDI-QTOF mass spectrometry

3.10.1. Chemicals and reagents

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

3.10.2. Solutions

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↓]

3.10.3. Special equipment

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

3.10.4. Procedure

3.10.4.1. Sample purification by nano-scale reversed-phase chromatography

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.

3.10.4.2. Protein ladder sequencing of peptide fragments

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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