Primary astroglial cultures were prepared from Sprague- Dawley rats (P1-2) as previously described (Nodin et al., 2005), with all experimental procedures approved by the Ethics Committee of the University of Göteborg. Briefly, rat pups were decapitated and the cerebral cortices were dissected and mechanically dissociated through 80-µm nylon meshes into Eagle’s minimum essential medium with 20% foetal calf serum (FCS, PAA Laboratories GmbH, Pasching, Austria), 2 mM L-glutamine and 1% penicillin/streptomycin (Life Technologies, Täby, Sweden). The medium had extra substances added to the following composition: 1.6 times the concentration of amino acids and 3.2 times the concentration of vitamins (Life Technologies), 48.5 mM NaHCO₃ and 7.15 mM glucose (Merck, Darmstadt, Germany). After three days in culture, the medium was exchanged with a customised Eagle’s minimum essential medium deficient in all amino acids with 1.6 times the concentration of amino acids lacking arginine and 3.2 times the concentration of vitamins (Invitrogen) added. The medium also contained 20% dialysed foetal calf serum (FCS, PAA Laboratories GmbH, Pasching, Austria), 2 mM L-glutamine, 1% penicillin/streptomycin (Life Technologies), 48.5 mM NaHCO₃ and 7.15 mM glucose (Merck). The cells were cultured in 5% CO₂ at 37ºC in media containing either normal ¹²C^{6 14}N₄-arginine (= 98.5% purity, Sigma Aldrich, St. Louis, MO) or isotopically labeled ¹³C₆ ¹⁵N₄-arginine (98% purity, Cambridge Isotope Laboratories, Andover, MA).

Confluent cultures (14-21 days after plating) were mechanically lesioned using a pipette tip in a 5 mm grid frame (Faijerson et al., 2006) and media were changed. Conditioned media were collected 48h after the injury was induced from both non-lesioned and lesioned astrocytes, filtered at 0.22 µm (Pall Corporation, East Hills, NY, USA) and immediately frozen at -80°C. After the conditioned media were collected, cells were harvested in ice-cold PBS supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland), 1 mM sodium orthovanadate (Sigma Aldrich) and 1 mM phenylmethanesulfonyl fluoride (Fluka, Sigma Aldrich). The samples were centrifuged (10 min, 4°C, 500 g), supernatants were discarded and pellets were stored at -80°C until usage. To validate the reactive astrogliosis model, cells were fixed (4% paraformaldehyde in PBS, 4°C, 10 min) 48h after the injury was induced and stained (1h, RT) with GFAP antiserum (rabbit, 1:500, DAKO, Glostrup, Denmark) in PBS containing 3% donkey serum (Jackson Immunoresearch Laboratories Inc., West Grove, PA) and 0.05% saponin (Sigma- Aldrich Sweden AB). Following three washes in PBS, cells were incubated for 1h at RT with the secondary antibody: Alexa Fluor 555-conjugated donkey anti-rabbit (1:2000, Molecular Probes) and the nuclear dye bisbenzimide from a stock at 5 ìg/ml (1:80, Hoechst 33258).

Conditioned media (200 g) from lesioned astrocytes, and from lesioned and non-lesioned astrocytes pooled in equal ratios, were protein precipitated by adding ice-cold acetone, three times the volume of the conditioned media. The samples were mixed, stored at -20°C overnight, centrifuged (10 min, 4ºC, 35,000 g), and the supernatants were discarded. Lysis buffer (9M Urea, 5% CHAPS and 0.05% SDS) supplemented with a protease inhibitor cocktail (Roche) was added to the cell pellets. The samples were sonicated (5x30 s), left in RT for 45 min and centrifuged (15 min, 4ºC, 35 000 g) before the supernatants were carefully removed. Protein concentrations were measured by the Bradford protein assay (BioRad, Hercules, CA, USA). Cell lysate (30 mg) from lesioned astrocytes, and from lesioned and non-lesioned astrocytes pooled in equal ratios, were treated with ReadyPrep (BioRad) according to the manufacturer’s instructions.

Protein pellets from conditioned media and cell lysates were dissolved in 25 mL of NuPAGE sample buffer (0.14 M Tris bas, 0.11 M Tris-HCl, 0.5 mM EDTA, pH 8.5, 10 % Glycerol, 75 mM LDS) and separation was performed on Nu- PAGE 10% Bis-Tris gels (Novex, San Diego, CA) and MOPS running buffer (50 mM MOPS, 50 mM tris, 3.5 mM SDS, 0.8 mM EDTA) at a constant voltage (200 V, 50 min). The gels were stained using Simply blue (Novex) to visualise the gel bands.

The gel lanes of the conditioned media with an apparent molecular weight below 45 kDa were excised and divided into 12 equal sized sections. The whole gel lanes of the cell lysates were divided into 20 equal sized sections. Gel pieces were reduced and alkylated prior to digestion (Amanchy et al., 2005). The peptides were extracted with a 1:1 mixture of NH₄HCO₃ (50 mM) and acetonitrile (ACN), followed by a 1:1 mixture of 5 % formic acid and ACN twice. The resulting supernatants were pooled, dried and stored at -20ºC until analysis.

The peptide samples from the conditioned media and the cell lysate were resolved in 15 and 25 μl of aqueous 0.1% formic acid, respectively. The liquid chromatography mass spectrometry (LC-MS) and LC-tandem MS (LC-MS/MS) spectra were acquired using a hybrid Linear Ion Trap Fourier Transform Ion Cyclotron Resonance mass spectrometer equipped with a 7 T magnet (LTQ-FT, Thermo Electron Corp., Bremen, Germany) coupled to a Ettan™ MDLC (GE Healthcare, Uppsala, Sweden) multi-dimensional nanoflow chromatography system. The Ettan™ MDLC controlled by UNICORN software was run in the high-through put mode with two parallel flow paths for desalting and separation of peptides prior to on-line MS and MS/MS analyses. A Zorbax 300 SB-C18 trap column, (5 mm, i.d. 0.3 mm, 5 ì, Agilent Technologies, Palo Alto, CA) was used for online desalting and sample cleanup, followed by a nano-scale reverse phase column (Zorbax 300 SB-C18, 150 mm, i.d. 0.075 mm, 3.5 ì, ,Agilent Technologies) for high-resolution separation. After 17 minute linear run, loading the precolumn, the separation was performed at a flow rate of approximately 200 nL/min by applying a linear gradient of 0–60% B for 50 min. Mobile phase A consisted of HPLC-grade water with 0.1% formic acid while mobile phase B was 84% HPLC grade aqueous ACN with 0.1% formic acid. The injected volumes were 5 ìl. Each sample was subjected to two independent analyses using two separate, but identical analytical columns. The eluent was electrosprayed (+1.3 kV) from the emitter tip (i.d 10 ìm, uncoated, pre-cut, PicoTip^TMEmitter, New Objective, Inc., Woburn, MA, USA) into the heated capillary of the mass spectrometer. The mass spectrometer operated in the data dependent mode to switch automatically between MS and MS/MS acquisition. In the first experiment of the conditioned media both MS and MS/MS scans were performed within the linear ion trap (LTQ) to maximise the sensitivity of the analysis. Each scan cycle consisted of one full scan mass spectrum (m/z 300–1500) collected in profile mode and the three most abundant doubly and triply charged protonated ions in each MS-scan were selected for isolation, fragmentation and detection in the LTQ. The survey MS spectra (m/z 400– 1600) of the conditioned media sample were also acquired with the Fourier Transform Ion Cyclotron Resonance (FTICR) MS followed by MS/MS of the three most abundant doubly and triply charged ions in the LTQ. Dynamic exclusion was activated during 30 s with a repeat count of 1 in the analysis of the conditioned media. In the experiment of the cell lysate the survey MS spectra (m/z 300–1500) were acquired with the FT-ICR MS followed by MS/MS of the three most abundant doubly and triply charged ions in the LTQ. Dynamic exclusion was activated during 60 s with a repeat count of 1. The resolution was set to 100 000 for the FT-ICR.

Acquired MS/MS data from the analysis of the conditioned media were submitted to database search using the inhouse Mascot software packet (Matrix Science, London, UK, (Perkins et al., 1999)). The data was searched against the NCBI database for protein identification. Database interrogation was; taxonomy (Mammals); enzyme (trypsin); variable modifications (carbamidomethyl, oxidation of methionine residues and 13C6 15N4-Arg); mass values (monoisotopic); protein mass (unrestricted); peptide mass tolerance (1 Da); fragment mass tolerance (±0.4 Da), peptide charge state (2+ and 3+) and max missed cleavages (1). The peptide mass tolerance was set to 20 ppm in the search of data acquired with FT-ICR cell and rodent was selected in the search of the cell lysates. All spectra from one gel lane, run on the same analytical column, were searched as merged files, resulting in two independent data sets per sample. Proteins considered as identified proteins had at least two peptides with an individual mascot score corresponding to p<0.05 and p<0.1, respectively. The proteins identified in the conditioned media originated from Rattus Norvegius. Random search of the LCMS/ MS analysis of the conditioned media resulted in an average of 51 000 queries for each merged search, and only one false positive identified proteins with the given criterion. For proteins quantitation DeCyder MS Differential Analaysis software (DeCyderMS, GE Healthcare (Johansson et al., 2006; Thorsell et al., 2007)) was used. Acquired LC-MS raw data were converted and the PepDetect module was used for automated peptide detection, charge state assignments, and quantitation based on the peptide ions signal intensities in MS mode. The ratios of the integrated signal intensities were calculated for all the detected charged states of the unlabeled and labeled peptides ions in the SILAC pairs.

The present study evaluated whether secreted proteins can be identified and differentiated from the medium proteins in serum-containing conditioned media by a combination of SILAC and mass spectrometry. Conditioned media from primary astrocytes and astrocytes in a scratch injury model of reactive astrogliosis substituted with either normal or isotopically labeled arginine served as our model system (Figure 1). The conditioned media samples were resolved by gel electrophoresis, revealing a distinct serum albumin protein band and a number of less intense protein bands (Figure 2). The gel lanes were excised and divided into equal sized sections, ingel digested followed by nano-scale LC prior to mass spectrometric analysis. This approach allowed for identification of proteins in the complex mixture of tryptic peptides and extensive separation of the proteins prior to proteolysis was not required, owing to the fact that peptides were isolated and fragmented individually in the mass spectrometric analysis.Table 1

Most of the proteins identified in the conditioned media were bovine proteins derived from the serum. However, the database search also resulted in 22 proteins identified as proteins originating from rattus norvegicus in the serum-containing conditioned media (Table 1). For 12 of these proteins, at least one labeled arginine containing peptide was found, conf i rming that they were derived from the astrocytes rather than from the culture medium. The secreted proteins were differentiated from the medium components by the 10 Da mass differences of their fragment ions in the mass spectra corresponding to the incorporated arginine label (Figure 3). These results were confirmed with a second mass spectrometric analysis of the conditioned media (Table 1). However, the high mass accuracy obtained on the LTQ combined with FTICR was gained at the expense of loss in sensitivity compared to the analysis by the LTQ alone.

The amino acid sequences of the proteins identified were compared with the sequences of the corresponding bovine proteins, in order to evaluate the possibility to discriminate astrocyte derived proteins from the serum proteins by their amino acid sequences. Only six of the identified proteins generated tryptic peptides detected in the mass spectrometric analysis with sequences different from the bovine proteins and a total of 17 unlabeled unique peptides were found for these proteins (Table 1). This is in contrast to the 32 peptides containing the arginine label found in the mass spectrometric analysis and the resulting 12 proteins that could be distinguished from the medium components. Thus, the incorporated arginine label was more effective in identifying secreted proteins in the serum-containing conditioned media than their amino acid sequences.

The released proteins in the conditioned media were also compared with the proteins identified in the whole cell extract. A number of proteins were identified in both the conditioned media and the cell lysate (Table 1). This finding strengthens the result that the proteins found in the conditioned media were derived from the astrocytes. Several of the proteins were only found in the conditioned media, suggesting that these proteins were enriched in the media. In the whole cell lysate, the level of these proteins was probably too low compared with the other relatively highly abundant proteins to allow for their identification.

The identified proteins in the serum-containing conditioned media included protease inhibitors (cystatin C), carrier proteins (insulin-like growth factor binding protein 2 (IGFBP- 2) and apolipoprotein E (ApoE)), antioxidants (Cu/Zn superoxide dismutase, peroxiredoxin 1, 4, 6), proteins involved in remodelling (clusterin, secreted protein acidic and rich in cysteine (SPARC)) and structural proteins (GFAP, vimentin, cofilin and beta-actin).

A similar profile of released proteins was found in the conditioned media from lesioned and non-lesioned astrocytes. Neither were any significant quantitative differences between the two cell cultures found. However, this finding was a confirmation that the proteins in the lesioned astrocyte conditioned medium were secreted, rather than being the result of intracellular proteins leaking from ruptured cells. The quantitative analysis of a protein in the pooled conditioned media was based on the peptide ion intensities of the unlabeled and labeled peptides in the SILAC pairs. In Figure 4 representative two-dimensional (2D) signal intensity maps of the LC-MS analysis used for relative quantitation and the LC-MS spectra of a peptide pair are shown. The 2D signal intensity map of the pooled sample illustrates that no quantitative differences could be found for the protein.

Our results also revealed inherent limitations in the quantitative analysis of lesioned and non-lesioned astrocyte conditioned media. Surprisingly, the analysis of the lesioned astrocyte conditioned medium revealed both unlabeled and labeled peptides with the same amino acid sequence for some of the identified proteins (Figure 3). In the lesioned astrocyte conditioned medium only the labeled arginine containing peptide should be detected. The presence of both peptides could either be due to an inefficient labeling of the cellular proteins or that the unlabeled tryptic peptides originate from the medium components. In order to determine the labeling efficiency, the whole cell lysate of the lesioned astrocytes was analysed. The analysis showed close to complete incorporation of the labeled amino acid into the proteins, since peptides containing the unlabeled amino acid were nearly absent in the MS-spectra. This finding confirmed that the unlabeled proteins were present in the culture medium itself and that several of the secreted proteins generated tryptic peptides with the same amino acid sequences as the corresponding bovine proteins. Consequently, the signal ion intensity from the unlabeled peptides in the pooled sample included both proteins released from the astrocytes and from the culture medium itself, which complicated the quantitative analysis for these proteins. To circumvent this drawback and to calculate a correction factor to obtain the correct expression ratios, the intensity ratios of both lesioned astrocyte conditioned medium and the pooled samples were calculated. An estimation of the contribution of the medium components to the unlabeled peptides in the SILAC pair was thereby obtained. However, despite these analyses no quantitative differences were observed for these proteins. Furthermore, co-elution of different peptides with close to the same m/z during the LC-run also restricted the quantitative analysis. Several of the SILAC pairs identified in the fragmentation analysis that could not be quantified had interfering peptides with similar physicochemical properties that eluted closely enough to overlap in the LC-separation (Figure 5). Moreover, the quantitative analysis was limited by the generally low number of detected SILAC peptide pairs. To be able to calculate statistically significant intensity ratios, it is essential to obtain multiple SILAC pairs from each protein.