Platelet membrane proteomics: a novel repository for functional research
Blood Lewandrowski et al.
114: e10
Supplemental materials for: Lewandrowski et al
Platelet purification
Use of platelet apheresis concentrates was approved by the local ethics committee and donors were informed upon use of concentrates for research purposes. The concentrates were randomly chosen from the donor collective and do not reflect a certain subpopulation, sex or health state. Briefly, fresh apheresis concentrates (leukocyte depleted, 2–4 × 1011 platelets/250 mL, Department of Transfusion Medicine, University Würzburg, Germany) were divided into 50 mL centrifugation tubes and spun twice at 310 g for 15 min to remove potentially remaining leucocytes or erythrocytes. The supernatant of the second centrifugation step was spun again at 380 g for 20 min at room temperature. To remove residual plasma proteins the resulting pellets were washed twice with 10 mM citric acid buffer containing 5 mM KCl, 145 mM NaCl, 14 mM glucose, and 1 mM MgCl2, pH 6.4. Final pellets were frozen in liquid nitrogen until further use.
Multidimensional protein identification technology
For MudPIT analysis, custom made triphasic columns were packed in 100 µm ID fused silica capillaries with a fritless emitter tip. Columns consisted of 3 cm C18 material (Luna, 3 µm particle size, Phenomenex, Aschaffenburg, Germany) followed by 5 cm SCX (polySULFOETHYL A, 5 µm particle size, Chromatographic Technologies, Basel, Switzerland) and a 10 cm C18 separation phase (Luna, 3 µm particle size, Phenomenex). Samples were applied to the column in 0.1% formic acid within a pressurized compartment. Thereafter, the column was connected to an Ultimate dual gradient system (Dionex, Idstein, Germany) and washed for 30 min at 300 nL/min flow with buffer A (0.1% formic acid, 2% acetonitrile). Subsequently, a first reversed phase gradient from 8% B to 60%B in 145 min was used to transfer peptides to the SCX phase. Buffer B was 0.1% formic acid, 80% acetonitrile. Stepwise elution and separation of salt-eluted peptides was achieved by applying increasing steps of buffer C (250 mM ammonium acetate, 0.1% formic acid) or buffer D (1.5 M ammonium acetate, 0.1% formic acid) prior a reversed phase gradient from 7% B to 60% B in 65 min. Salt steps were 10/20/30/40/50/60/70/80/90/100% C and 33/66%D for 2 min.
Mass spectrometric detection was performed using a LTQ XL collecting a single full MS spectrum for m/z 420–2000 followed by three MS/MS spectra with a normalized collision energy of 35%. Dynamic exclusion was set to 30 s with a 3 Da window for each fragmented ion signal. Spray voltage was set to 1.8 kV.
Methionine COFRADIC
The membrane platelet pellet was lysed with 2 M GuHCl in 50 mM sodium phosphate buffer (pH 8.0). Disulfide bonds were reduced by addition of 10 mM DTT and incubation for 30 min at 56°C. Carbamidomethylation was performed with 5 mM IAA and incubation for 30 min at room temperature in the dark. Subsequently, the sample was diluted 1:10 (v/v) with digestion buffer (50 mM ammonium bicarbonate). Tryptic digestion was performed at 37°C overnight (protease to protein ratio 1:20 (w/w)). Resulting peptides were purified by C18 solid phase extraction columns (SPEC 3 ml C18AR 30 mg, Varian), lyophilized, resuspended in 0.1% trifluoroacetic acid and separated by a primary RP-HPLC run (Gradient see below table 1). In total 48 fractions of one minute duration were collected during the gradient, starting at 10% B. Fractions were pooled (see table 2) and methionine residues were oxidized prior to the secondary run for 30 min at 30°C by adding H2O2 to a final concentration of 0.5% (v/v). Each fraction was then separated by a secondary RP-HPLC run, using identical chromatographic conditions as in the primary one. Due to the hydrophilic shift, methionine containing peptides elute roughly 1–8 min earlier than in the primary run. The collected fractions were dried and prepared for LC-MS/MS analysis.
Cysteine COFRADIC
The membrane platelet pellet was lysed with 2M GuHCl in 50 mM sodium phosphate buffer (pH 8.0). Disulfide bonds were reduced by addition of 10 mM DTT for 30 min at 56°C. Next, the sample was desalted by a NAP-5 column (GE Healthcare), while lysis buffer was used for elution. The sample was reduced to complete dryness by vacuum drying and reconstituted again in 10 mM Ellman’s reagent, 100 mM Tris (pH 8.7) and incubated for 1 h at 37°C. Once again the sample was desalted by a NAP-5 column and eluted with 10 mM Tris (pH 8.7). Tryptic digestion was performed at 37°C overnight (protease to protein ratio 1:20 (w/w)).
After proteolytic digest, peptides were purified by C18 solid phase extraction columns (Varian) and lyophilized. The sample was reconstituted in 0.1% trifluoroacetic acid, H2O2 was added to a final concentration of 0.5% (v/v) followed by incubation for 30 min at 30°C.
The digest was separated by a primary RP-HPLC run (Gradient see table 1). In total 48 fractions of one minute each were collected during the gradient, starting at 10% B. Fractions were pooled (see table 3) to a final number of 16 fractions. Fractions were dried under vacuum and reconstituted in 10 mM Tris, 50 mM DTT and incubated for 30 min at 56°C. The reaction was stopped by adding 0.1% trifluoroacetic acid (till pH 2). All fractions were applied to a secondary RP-HLC run, using identical chromatographic conditions as in the primary one. Fractions were recollected in a time interval 3 to 10 min before the elution time in the primary run. Collected fractions were dried and prepared for LC-MS/MS analysis.
N-terminal COFRADIC
The membrane platelet pellet was lysed with 2M GuHCl in 50 mM sodium phosphate buffer (pH 8.0). Disulfide bonds were reduced by addition of 10 mM DTT and incubation for 30 min at 56°C. Carbamidomethylation was performed with 5 mM IAA and incubation for 30 min at room temperature in the dark. Acetylation of free amines was performed with 25 mM deutero-acetyl N-Hydroxy-Succinimide, for 1 h at 37°C. Acetylation of serine and threonine was reversed by adding hydroxylamine in fourfold excess of deutero-acetyl N-Hydroxy-Succinimide. The reaction was stopped by adding glycine in twofold excess to deutero-acetyl N-Hydroxy-Succinimide. Afterwards the sample was diluted 1:10 with digestion buffer (50 mM ammonium bicarbonate). Tryptic digestion was performed at 37°C overnight (protease to protein ratio 1:20 (w/w)).
After the digestion step, peptides were purified by C18 solid phase extraction columns (Varian) and lyophilised. Afterwards the sample was reconstituted in 0.1% trifluoroacetic acid, H2O2 was added to a final concentration of 0.5% (v/v) followed by incubation for 30 min at 30°C. Subsequently, the sample was separated in a primary RP-HPLC run (Gradient see table 1). In total 16 fractions of four minute each were collected during the gradient, starting at 10% B. Fractions were dried under vacuum and reconstituted in 50 µl 50 mM sodium borate (pH 9.5). Then 10 µl 15 mM TNBS were added and incubated for 30 min at 37 °C; this step was repeated three times. The reaction was stopped by adding 20 µl 10% trifluoroacetic acid. All fractions were applied to a secondary RP-HLC run, using identical chromatographic conditions as in the primary one. Fractions were recollected from 4 min before to 4 min after the initial elution time in the primary run. Collected fractions were dried and prepared for LC-MS/MS analysis.
COFRADIC reversed-phase separation
COFRADIC primary and secondary separations were performed on an Ultimate 3000 liquid chromatography system consisting of a LPG 3000 quaternary micro pump, a TCC 3400 column compartment, an UVD 3400 variable wavelength detector (run at 214 nm with a 180 nL flow-cell) and a WPS-T 3000 well plate sampler equipped with 8-port valve, 0.15 mm bore for fractionation option, with a 125 µl loop (all Dionex, Idstein, Germany). For reversed-phase separation a Zorbax 300SB-C18 column (5 µm particle size, 2.1 × 150 mm, Agilent) was used at a flow rate of 80 µL/min and 30°C. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.08% trifluoroacetic acid in 84% acetonitrile.
