Cells
Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coat preparations by Ficoll density gradient centrifugation. For the generation of PHA-stimulated lymphoblasts, T cells were purified by magnetic cell sorting (MACS) using cell isolation kits from Miltenyi Biotech (Bergisch Gladbach, Germany). The cells were stimulated with phytohemagglutinin A (PHA, 0.5 μg/ml, Remel, Lenexa, KS, USA) and expanded in the presence of irradiated EBV-transformed B cells and allogenic PBMC and subsequently with recombinant interleukin 2 (rIL-2, 100 U/ml, Chiron GmbH, Marburg, Germany). Before the cells were analyzed on day 12-14, dead cells were removed by Ficoll-gradient centrifugation resulting in a > 98% pure T cell population as judged by CD3 FACS analysis.
Confocal microscopy
Cells were fixed with 3% paraformaldehyde and permeabilized with 1% Triton X-100 as described [13]. The following antibodies were used: mouse IgG1 isotype-control MOPC-21 (Abcam, Cambridge, UK), anti-FasL mAb NOK1 (BD Biosciences, Heidelberg, Germany) with AlexaFluor488-conjugated goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany), anti-CD63 mAb clone MEM-259 (Immunotools, Friesoythe, Germany) conjugated to AlexaFluor555 (Invitrogen), anti-Granzyme A-FITC (Immunotools) and anti-Cathepsin B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with AlexaFluor488-conjugated donkey anti-goat IgG (Invitrogen). Stained samples were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and analyzed on a laser scanning microscope (LSM 510 Meta, Carl Zeiss, Jena, Germany) with appropriate filter settings. Images were acquired via scanning through the x-y-plane with 63 × objective lense. Laser intensity and detectors were adjusted to a uniformly negative signal of the control samples stained with control IgG and second step antibodies.
Subcellular fractionation
For subcellular fractionation and enrichment of secretory lysosomes, at least 4x108 T cells were used. The fractionation procedure has been recently described in detail [11]. Briefly, the cells were mechanically disrupted and organelles were enriched by differential centrifugation steps. The enriched organelles were then loaded on a discontinuous density gradient (4.4 ml volume) with 27%, 22.5%, 19%, 16%, 12%, 8% Optiprep® which is a 60% Iodixanol solution (Sigma, Deisenhofen, Germany) and subjected to ultracentrifugation. Interphases were collected from the top of the gradient resulting in six 400 μl fractions named and numbered 1 to 6. The protein content in each fraction was determined using a Coomassie Protein Assay Reagent (Thermo, Rockford, IL, USA).
Western blot analysis
For Western blotting, 5 μg of protein were separated by SDS-PAGE on pre-casted 4-12% gradient Bis-Tris gels (Invitrogen). After transfer to nitrocellulose (NC) membranes (Biometra, Goettingen, Germany) and blocking with 5% BSA or dry milk, the fractions were analyzed for subcellular marker proteins with the following antibodies: anti-FasL clone G-247.4 (BD Biosciences), anti-CD63 clone MEM-259 (Acris Antibodies, Herford, Germany), anti-LAMP-1 clone 25 (BD Biosciences), anti-cathepsin D clone CTD-19 (Sigma), anti-cytochrome oxidase IV (CoxIV) mAb clone 10G8D12C12 (1/1000, MitoScience, Eugene, OR, USA), anti pan-cadherin clone ab22744 (Abcam, Cambridge, UK) and horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (GE Healthcare, Munich, Germany). Membranes were prepared for reprobing by incubation in stripping solution (100 mM 2-mercaptoethanol, 2% SDS, 60 mM Tris) for 25 min at 56°C. ECL reagents in combination with Hyper Film (GE Healthcare) were used for chemiluminescence detection.
Transmission electron microscopy
Enriched organelles of fractions 2 and 5 were fixed with a mixture of 3% paraformaldehyde and 0.05% glutaraldehyde in PBS at 4°C overnight, washed in PBS, postfixed in 2% OsO4, dehydrated in ethanol, and embedded in araldite (Sigma, Deisenhofen, Germany). Ultrathin sections were mounted on formvar-coated grids and double-stained with a saturated solution of uranyl acetate in 70% methanol and lead citrate. The grids were examined with a Zeiss EM 900 transmission electron microscope equipped with a digital camera system.
2D electrophoresis, image analysis and spot picking
The 2D electrophoresis was performed as described before [11]. Briefly, SL pellets of fraction 2 were lysed on ice for 30 min with 30 μl lysis buffer (pH 8.5) containing 7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS. The supernatant was recovered after centrifugation for 20 min at 20.000 × g at 4°C. A total amount of 250 μg of protein was mixed with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% (v/v), IPG buffer pH 3-11 and 2% (w/v) DTT) and applied by cup-loading onto 24 cm non-linear pH 3-11 IPG gel strips for isoelectric focusing (IEF). The second dimension was performed on 26 × 20 cm large 12.5% polyacrylamide gels after reduction and alkylation using the Ettan DALTsix large vertical electrophoresis system from GE Healthcare. The gels were removed from the glass plates, stained with Flamingo Pink (Bio Rad), mounted on a non-backed gel frame, scanned on a Typhoon Trio imager (GE Healthcare) and analyzed using Image Master 6.0 (GE Healthcare). Selected spots were picked with a 2 mm picking head. The picked gels were again scanned to verify the correct location of the punched spots.
In-gel tryptic digestion and mass spectrometry
Gel plugs were washed with water and 12.5 mM ammonium bicarbonate (ABC) in 50% acetonitrile (ACN) and dehydrated in pure ACN. The dry gel pieces were rehydrated with 100 ng sequencing-grade trypsin (Serva, Heidelberg, Germany) in 5 mM ABC and tryptic in-gel digestion was performed at 37°C overnight. For peptide extraction, 0.3% trifluoroacetic acid (TFA) in ACN was added and the samples were sonicated for 15 min. The liquid phases were collected, lyophilized, redissolved in 0.5 to 1 μl MALDI matrix solution (3.2 mg/ml α-cyanohydroxycinnamic acid (Sigma) in 65% ACN/0.1% TFA), spotted onto 192-well stainless steel MALDI plates and air-dried. The samples were analyzed by peptide mass finger printing in positive reflectron mode followed by MSMS analyses of the most apparent five peptides using the 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, Framingham, MA, USA) as described elsewhere [12]. Peptide mass spectra were processed by internal calibration with autolytic fragments of porcine trypsin with 25 ppm mass tolerance. MSMS spectra were acquired using default calibration updated prior to the run. Spectral data were searched against human proteins in the NCBI database (Homo sapiens, 192,176 entries) using MASCOT V2.0 (Matrix Sciences, London, UK).
Database analysis
Database searches with MASCOT were performed using the following parameters: the modification on cysteine residues by carbamidomethylation was set as obligate, methionine oxidation was considered as a potential modification; the maximum number of missed tryptic cleavages was one; the monoisotopic masses were considered and the mass tolerance was set to ± 50 ppm, and the fragment-ion mass tolerance was set to 0.2 Da (MS/MS). A protein was accepted to be identified when the total protein score reached or exceeded the MASCOT score threshold (≥ 65 with a probability of identification greater 95%). A repeated search against a randomized decoy database (http://www.matrixscience.com/help/decoy_help.html) using the decoy.pl script and identical search parameters let to a false-positive rate of 1.2%.
The classification according the localization and function of individual proteins was based on the Uni-Prot knowledge base, the iHOP database [19] and the iProXpress database [17] available through the Protein Information Resource (PIR) (GUMC, Washington DC, USA). Identified proteins were searched in this organelle-proteome reference dataset according to their Uni-Prot numbers.