The rat SCs were harvested as previously described  with minor modifications. Briefly, sciatic nerves were harvested from Sprague-Dawley rats (1 to 3 d- old) and enzymatically dissociated by incubation at 37°C sequentially with 1% collagenase and 0.125% trypsin for 30 and 10 min, respectively. The mixture was triturated, centrifuged and resuspended in 10% FBS in DMEM. The cell pellets were plated on poly-L-lysine precoated dishes (35 mm) for incubation in the same medium. On the following day, 10 μM cytosine arabinoside was added and allowed to incubate for an additional 48 h to remove fibroblasts. The cell culture was maintained subsequently in DMEM supplemented with 10% FBS, 2 μM forskolin (Sigma, St Louis, MO) and 2 ng/ml heregulin (HRG, Sigma) to stimulate SC proliferation. For further purification, the cell culture was gently trypsinized, pelleted, and incubated with anti-Thy1 antibody (AbD Serotec, Raleigh, NC) on ice for 2 h, followed by incubation in complement (Jackson Immuno, West Grove, PA) for an additional 2 h. All media and supplements were bought from Gibco-Invitrogen (Carlsbad, CA).
After isolation and purification, primary cultured SCs were subjected to immunocytochemistry with anti-S100β, anti-GFAP, anti-TUBB3, anti-NEFM or anti-ATG5. Briefly, the cell culture was fixed in 4% paraformaldehyde (pH 7.4) for 30 min, permeabilized with 0.3% Triton X-100, 10% goat serum in 0.01 M phosphate buffered saline (pH 7.2) for 60 min at 37°C, and allowed to incubate with anti-S100β, anti-GFAP (1:200, Abcam, Cambridge, MA), anti-ATG5 (1:200, Abgent INC, San Diego, CA) and anti-TUBB3, anti-NEFM (1:200, Sigma) antibody respectively at 4°C overnight, followed by reaction with FITC- or PE-conjugated goat anti-rabbit IgG (1:400, Molecular probes, the Netherlands) for 2 h at room temperature, respectively. The cells were also stained with 5 μg/ml Hoechst 33342 dye at 37°C for 10 min. The fluorescence was visualized under a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).
For flow cytometric analysis (FCA), primary cultured SCs were dissociated by treatment with 0.125% (w/v) trypsin, and the cell pellets were resuspended in a fixation medium (Invitrogen, Carlsbad, CA) and incubated for 15 min at room temperature. Permeabilization Medium (Invitrogen, Carlsbad, CA) and the recommended volume of the anti-S100β antibody were added (Abcam, Cambridge, MA) to allow incubation for 20 min. Cells were then stained with CFTM488A IgG secondary antibodies (Biotium, Hayward, California) at room temperature for 30 min. Flow cytometric acquisition and data analysis were performed with a flow cytometer and cellquest software (BD FACScalibur, BD Bioscience, San Jose, CA). As a negative control, the cells were incubated only with the FITC-conjugated secondary antibody. Three independent flow cytometric experiments were performed.
Cell cultures were washed with ice-cold phosphate buffered saline (PBS) and lysed in a buffer containing 50 mM Tris-HCl (pH7.6), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 0.1 mM Na3VO4, 1% (v/v) Triton X-100, 1 mM PMSF, and a protease inhibitor mixture tablet (Roche Applied Science, Mannheim, Germany). Lysates were clarified by centrifugation at 15,000 × g for 20 min at 4°C, and protein concentration was determined by Bradford protein assay (Bio-Rad, Richmond, CA).
Digestion, sample cleaning, and desalting
Protein from primary cultured SCs was precipitated with ice-cold acetone overnight at -20°C, and pellets were dissolved, denatured, alkylated and digested with trypsin (1:20, w/w, Sigma) at 37°C for 18 h. Prior to on-line 2D nano LC/MS/MS analysis, samples were cleaned and desalted. A cation exchange cartridge system (Applied Biosystems, Foster city, CA) was used to remove the reducing reagent, SDS, undigested proteins, and trypsin in the sample mixture because these materials would interfere with the LC/MS/MS analysis. Subsequently, the eluate of cation exchange was desalted on a 4.6-mm-inner diameter × 150-mm C18 reversed-phase column (5 μm, 80 Å; Agilent, Waldbronn, Germany).
On-line 2D nano LC/MS/MS
2D nano LC/MS/MS analyses were conducted on a nano-HPLC system (Agilent, Waldbronn, Germany) coupled to a hybrid Q-TOF mass spectrometer (QSTAR XL, Applied Biosystems) equipped with a nano-ESI source (Applied Biosystems) and a nano-ESI needle (Picotip, FS360-50-20; New Objective Inc., Woburn, MA). Analyst™ 1.1 software was used to control the QSTAR XL mass spectrometry and nano-HPLC system and to acquire mass spectra. Vacuum-dried peptides were reconstituted in phase A and injected at a flow rate of 10 μl/min onto a high resolution strong cation exchange (SCX) column (Bio-SCX, 300-μm inner diameter × 35 mm; Agilent, Wilmington, DE), which was on line with a C18 precolumn (PepMap, 300-μm inner diameter × 5 mm; LC Packings). After loading, the SCX column and C18 precolumn were flushed with a 16-step gradient sodium chloride solution (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 300, and 400 mM) for 5 min and phase A for 10 min at a flow rate of 15 μl/min. Afterwards, the precolumn was switched on line with a nanoflow reversed-phase column (VYDAC 218MS, 75-μm inner diameter × 100 mm; Grace, Hesperia, CA), and the peptides concentrated and desalted on the precolumn were separated using a 120-min linear gradient from 12 to 30% (v/v) phase B (0.1% (v/v) FA in acetonitrile) at a flow rate of 400 nl/min.
The Q-TOF instrument was operated in a positive ion mode with ion spray voltage typically maintained at 2.0 kV. A mass spectrum of the sample was acquired in an information-dependent acquisition mode. The analytical cycle consisted of a 0.7-s MS survey scan (400-1600 m/z) followed by three 2-s MS/MS scans (100-2000 m/z) of the three most abundant peaks (i.e. precursor ions), which were selected from the MS survey scan. Precursor ion selection was based upon ion intensity (peptide signal intensity above 25 counts/s) and charge state (2+ to 4+), and once the ions were fragmented in the MS/MS scan, they were allowed one repetition before a dynamic exclusion for a period of 120 s. Under collision-induced dissociation (CID), fragment ions of the peptides were produced, resulting in sequencing of the peptides and identification of the corresponding proteins. External calibration of mass spectrometer was carried out routinely using reserpine and trypsinized bovine serum albumin.
The complete set of raw data files (*.wiff) of each run were uploaded to ProteinPilot software 3.0 (Applied Biosystems) and searched against the non-redundant International Protein Index (IPI) rat sequence database (version 3.62, 39,867 entries). The search parameters were as follows: trypsin digestion; methyl methane thiosulfate alkylation of cysteine residue, instrument, QSTAR ESI; identification focus, biological modifications, and FDR analysis selected. ProteinPilot uses Paragon algorithm for peptide identification and ProGroup algorithm to assemble the peptide evidence from the Paragon algorithm to find the smallest number of proteins that could explain all the fragmentation spectral evidence. Protein identification is based on the Unused ProtScore score, which is a measurement of all the peptide evidence for a protein that is not better used by a higher-ranking protein. In this study, the identification of a protein was reported for unique peptides with an "unused" confidence threshold (ProtScore) of > 1.3%, and with a corresponding FDR of less than 1%.
Functional category and localization of identified proteins
To obtain an overview of their biological significance, the identified proteins were categorized according to their main biological functions collected from the Uniprot protein knowledge database and PubMed. The localization of the proteins was analyzed by Ingenuity pathway analysis (IPA, http://www.ingenuity.com).
Total RNA was extracted from the primary cultured SCs using Trizol (Invitrogen), and cDNA was synthesized from the total RNA using the SuperScript First-Strand Synthesis System (Invitrogen). The qPCR was conducted by FastStart® SYBR Green qPCR Master Mix (Roche, Germany) according to the manufacture's specifications. A 50-μl reaction consisted of 1 μl of cDNA, 25 μl of 2 × Fast SYBR Green Master Mix, 1 μl of each primer, and 22 μl of RNase/DNase free water. Two-step fast cycling protocol was used in StepOne™ Real-Time PCR System (Applied Biosystems), and the data were analyzed using the software supplied by the vendor (Applied Biosystems). Primer sequences are reported in Additional file 2.
Western blot analysis
Cell proteins were extracted from primary cultured SCs and quantified by a BCA kit. Samples containing 15 μg of total protein were separated by 12% (w/v) SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA). After incubation for 1 h in 5% (w/v) nonfat milk in TBS-T buffer, the membrane was probed with the indicated primary antibodies overnight at 4°C. After wash with TBS-T, the membrane was incubated with IRDye 800-Conjugated secondary antibodies (Odyssey) for 1 h at room temperature. The images were scanned with the GS800 densitometer scanner (Bio-Rad, Hercules, CA). The primary antibodies used were rabbit polyclonal antibody against CNP or GFAP (Bioworld Technology Inc. Louis Park, MN), rabbit polyclonal antibody against NGFR (Abcam UK), goat polyclonal antibody against TUBB 3, mouse monoclonal antibody against NEFM (Sigma), and rabbit polyclonal antibody against ATG5 (Abgent, San Diego, CA).
Data are presented as the mean ± S.D. Data comparison was performed by unpaired Student's t-test with SPSS10.0 software. Statistical significance was set at p < 0.05. Unless otherwise specified, all assays were conducted at least in triplicate.