A proteome map of primary cultured rat Schwann cells

Background Schwann cells (SCs) are the principal glial cells of the peripheral nervous system with a wide range of biological functions. SCs play a key role in peripheral nerve regeneration and are involved in several hereditary peripheral neuropathies. The objective of this study was to gain new insight into the whole protein composition of SCs. Results Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS) was performed to identify the protein expressions in primary cultured SCs of rats. We identified a total of 1,232 proteins, which were categorized into 20 functional classes. We also used quantitative real time RT-PCR and Western blot analysis to validate some of proteomics-identified proteins. Conclusion We showed for the first time the proteome map of SCs. Our data could serve as a reference library to provide basic information for understanding SC biology.


Background
Schwann cells (SCs) are the principal glial cells of the peripheral nervous system (PNS) with a wide range of biological functions [1]. Basically, SCs are able to wrap around the axons of neurons to form compact myelin sheaths, which allow for rapid and saltatory conduction of electrical impulses and support the integrity of axons in the PNS. SCs may also perform functions other than myelin formation; for example, SCs are involved in trophic support for neurons, formation of the neural extracellular matrix, modulation of neuromuscular synaptic activity, and orchestration of inflammation in the PNS. Collectively, SCs play a key role in the normal development and function of the PNS.
After peripheral nerve injury, SCs aid in cleaning up the tissue debris and guide the regrowth of axons. To accomplish this, SCs proliferate to form longitudinal cell strands known as bands of Bungner, release neurotrophins, and guide the regenerating axons to target organs. On the other hand, several hereditary peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT), Guillain-Barré syndrome (GBS), schwannomatosis, and chronic inflammatory demyelinating polyneuropathy (CIDP), are probably caused by genetic mutations in SCs, a knowledge of which is required for the prevention and treatment of these neuropathies.
Collectively, SC biology has been an active area of neuroscience research. As compared to genomic and transcriptomic analysis, the identification of the protein composition of SCs might be more valuable in examining the biological characteristics of SCs. Despite some subcellular studies on the proteomic profile of SC mitochondria in the disease state [2], there are few studies dealing with the comprehensive analysis of cellular proteins in SCs. In this study, therefore, we aimed to establish a proteome map of primary cultured SCs.
Proteomics strategy represents a powerful tool in the global investigation of a great multitude of cellular proteins, but the strategy has relied previously on twodimensional gel electrophoresis (2-DE), which suffers from several drawbacks, including labor intensity, limited dynamic range, and an inability to detect hydrophobic, alkaline, and low abundance proteins under standard conditions [3,4]. The gel-based proteomics strategies are being replaced rapidly by a new proteomics strategy based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) [5][6][7][8], which shows a high degree of specificity and sensitivity.
By using 2D LC-MS/MS analysis, we identified a total of 1,232 proteins from primary cultured SCs and accomplished functional classification of the identified proteins. Obviously, the new insight into the protein composition of SCs not only contributes to the understanding of SC biology, but also provides an important basis for comparative studies between normal and diseased SCs.

Isolation and characterization of primary cultured SCs
For isolation and purification of SCs in vitro, we adopted efficient procedures to eliminate contamination of fibroblasts. The light micrograph demonstrated the typical morphology of primary cultured SCs ( Figure 1A). The purity of primary cultured SCs was confirmed by flow cytometry data ( Figure 1B), which indicated that 98.56% of the cell population was S100 β-positive (S100 β protein serving as a SCs marker). Immunocytochemistry with anti-S100 β and anti-GFAP provided further evidence for cell purity ( Figure 1C and 1D).

Identification, functional category, and subcelluar localization of cellular proteins in SCs
According to the criteria for protein identification, as mentioned in Materials and Methods, more than 700 proteins were identified in each independent biological replicate, and the corresponding false discovery rates (FDRs) of three individual analyses were less than 1%. Detailed information on identified peptides and proteins is provided in Additional file 1. Subsequently, proteins identified from three independent analyses and proteins under identical accession number and/or gene symbol were merged. The total number of proteins identified in this study was 1,232. Out of 1,232 proteins, 846 were identified by two or more unique peptides and the remaining 386 were identified by one unique peptide. The annotated spectra of proteins identified on the basis of one unique peptide are provided in Additional file 1. The Venn diagram ( Figure 2) shows that among 1,232 identified proteins, 555 (45%) were shared by all three experiments, and 271 (22%) were shared by two experiments ( Figure 2); thus, 67% of the proteins were identified by more than one experiment, confirming the good reproducibility of the adopted proteomics platform in protein identification. Furthermore, the 1,232 identified proteins were categorized into 20 different classes in terms of their main biological functions by searching the UniProt protein knowledge database http://www.uniprot. org/ and PubMed http://www.ncbi.nlm.nih.gov/pubmed/ ( Figure 3A), and the details are provided in Additional file 1. The identified proteins were further assigned to various subcellular compartments of SCs using ingenuity pathway analysis (IPA) software ( Figure 3B). The results of subcelluar localization of the 1,232 proteins are listed in Additional file 1. It was noted that almost half of the proteins were located in the cell cytoplasm.

Cell culture
The rat SCs were harvested as previously described [17] 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).

Flow cytometry
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.

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-mminner diameter × 150-mm C 18 reversed-phase column (5 μm, 80 Å; Agilent, Waldbronn, Germany).
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.

Protein identification
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   (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).

Statistical analysis
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.

Discussion
There have been several proteomic studies concerning the central nervous system (CNS), providing us with considerable information in the field of neuroproteomics [18][19][20][21][22][23][24][25][26][27][28]. In contrast, SCs, as one of the key components in the PNS, have received little research attention from the perspective of proteomics. A pioneer study accomplished proteome analysis of PNS myelin, which, formed by SCs, is comprised of multiple compacted layers of the molecularly specialized plasma membrane extended from SCs [29]. This study focused on the large-scale proteomics analysis of SCs themselves, which has hardly been reported before.
Metabolism-related proteins represented the largest class (22.8%) of proteins expressed in SCs. For example, APOE (apolipoproteinE), a lipid metabolism-related protein, has been shown to protect mice from chronic inflammatory demyelinating polyneuropathy by affecting the antigen-presenting function of SCs [11], and to benefit axonal reconstruction and myelin membranes [30]. It follows that this type of protein is responsible for some of the biological characteristics of SCs.
Intriguingly, autophagy and autophagy-related proteins were expressed in SCs. Autophagy, as a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents, is generally activated by conditions of nutrient deprivation, and is also associated with a diverse array of physiological and pathological processes, including development, differentiation, infection, cancer and neurodegeneration [31]. The identification of conserved autophagy genes (ATGs) suggests the existence of autophagy [32], and autophagy upregulation may assist in curing diseases caused by toxic intracellular aggregate-prone proteins or may serve as a lifespan extender in the normal body [33]. Previous studies have shown that glial cells from neuropathic mice activate autophagy in response to rapamycin and produce abundant myelin internodes, and that both the ubiquitin-proteasome system and autophagy mediate the traumainduced axonal degeneration and the retrieval of nerve growth factors [34,35]. In this study, we detected a class of autophagy-related proteins, including ATG3, ATG5, LAMP1, and UBQLN1, in primary cultured SCs. As is known, ATG5 protein contributes to inhibiting lethal Sindbis virus infections of the CNS in mice [36] and is involved in immune-mediated myelin injury in mice [37]; disruption of autophagy by mutation of ATG5 or ATG7 causes neurodegeneration [38]. In our study, qPCR, Western blot and immunohistochemistry confirmed the expression of the ATG5 protein in primary cultured SCs. Therefore, we assumed that the expressions of ATG5 and other autophagy-related proteins in SCs might influence the PNS. Another interesting finding of our confirmatory tests is that some proteins generally considered to be expressed in neurons, such as TUBB3 (tubulin3 β chain), TUBB2 (tubulin2 β chain), and NEFM (neurofilament medium polypeptide), were also present in SCs. Previous studies have reported the mRNA expression of NEFM and tubulin in SCs and TUBB3 expression in neurofibroma SCs [39,40]. To a certain degree, our results provided further evidence for these previous studies.

Conclusion
In this study, the 2D LC/MS/MS approach illustrated the proteome map of primary cultured SCs. We identified a total of 1,232 proteins, among which, 846 were identified by two or more unique peptides while the remaining 386 were detected by one unique peptide. Our data could be used as a reference library to provide basic information for studying SC biology.

Additional material
Additional file 1: The protein list of three independent experiments, biological function categories and subcellular localization of 1,232 proteins, and annotated spectra of proteins identified on the basis of one unique peptide spectrum. Unused (ProtScore): a measurement of the protein confidence for a detected protein, calculated from the peptide confidence for peptides from spectra that have not already been completely "used" by higher scoring winning proteins. Total (ProtScore): a measure of the total amount of evidence for a detected protein. % Cov (95): The percentage of matching amino acids from identified peptides having confidence greater than or equal to 95 divided by the total number of amino acids in the sequence. (From Proteinpilot online help).
Additional file 2: The 22 primer sequences used in qPCR. and obtained grant funding. FD contributed to the concept and design of the project, manuscript preparation, and obtained grant funding. All authors have read and approved the final manuscript.