Protein expression of sensory and motor nerves Two-dimensional gel electrophoresis and mass spectrometry ☆

The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product (gi|55628), glyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylglutathione lyase, adenylate kinase isozyme 1, two unnamed proteins products (gi|55628 and gi|1334163), and poly(rC)-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.


INTRODUCTION
Peripheral nerves are nervous system structures that link sensory and motor terminals to the central nervous system. The nerves comprise sensory and/or motor axons associated with particular glial cell types, such as Schwann cells, in connective tissue [1] . Functional recovery of peripheral nerve lesions is dependent on accurate axonal regeneration to original target end-organs, and misdirection of regenerating axons leads to poor results following nerve injury and repair [2] . Preferential regeneration of motor and sensory peripheral nerves is influenced by respective end-organs of muscle and skin [3][4] . Schwann cells also express distinct sensory and motor phenotypes associated with regeneration in a phenotype-specific manner [5] . To date, the exact molecular mechanisms by which sensory and motor nerves differentiate remain poorly understood. The identification of these molecules could facilitate specific functional reconstruction of sensory and motor nerves. The rat femoral nerve bifurcates below the inguinal ligament into a motor branch, which leads to the quadriceps muscle, and a terminal sensory branch, which is the saphenous nerve [6] . These two branches are the typical motor and sensory nerves utilized in peripheral nerve research. The motor branch has been utilized as a source of motor nerve grafts, and the sensory branch has been harvested for sensory grafts. In addition, dorsal and ventral spinal roots contain different types of axons. Dorsal roots of spinal nerves contain central arms of pseudounipolar neurons, and these bodies are located in dorsal root ganglia. Axonal peripheral arms are clustered within the peripheral nerves, and ventral roots consist predominantly of efferent axons (somatic and autonomic motor), and the bodies are present in the ventral horn of spinal cord gray matter. Therefore, dorsal and ventral roots are ideal sources of relatively pure sensory and motor axons. Quantitative and qualitative assessment of the proteome is achieved by two-dimensional gel electrophoresis (2DGE), which generates a high-resolution, quantitative protein expression map; mass spectrometry is used to identify regulated proteins [7][8] . These procedures have been further modified for the identification of www.nrronline.org peripheral nerve proteins [9] . The present study was designed to analyze differentially expressed proteins between the motor branch of the femoral nerve and saphenous nerve, as well as between ventral and dorsal roots, using the European Molecular Biology Laboratory silver staining and modified nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry (NanoUPLC-nano-ESI-MS/MS). (Figure 1) A total of three 2DGE maps with good separation and clear protein spots were obtained from motor branches of the femoral nerve, saphenous nerves, ventral roots, and dorsal roots. ImageMaster 2D Platinum analysis revealed 1 010.67 ± 80.98, 1 122.67 ± 101.93, 972.33 ± 54.56, and 859.33 ± 96.67 protein spots in maps of motor branches of the femoral nerve, saphenous nerves, ventral roots, and dorsal roots, respectively, with a mean intragroup matching rate > 80%. A total of 13 protein spots exhibited significant differential expression between motor branches of the femoral nerve and saphenous nerves, and values were distributed from 5.0 to 9.0 (isoelectric point) and 20.0 to 60.0 kDa (molecular mass). Compared to motor branches of the femoral nerve, seven spots were upregulated, four spots were downregulated, one spot was specificially expressed, and one spot was not expressed in saphenous nerve tissues ( Figure 1). In addition, 16 protein spots exhibited significant differential expression between ventral and dorsal roots, and values were distributed from 4.0 to 9.0 for isoelectric point and 10.0 kDa to 60.0 kDa for molecular mass. Compared to ventral roots, six spots were upregulated, seven spots were downregulated, and three spots were not expressed in the dorsal roots ( Figure 2). The 29 protein spots with significant differential expression were selected for NanoUPLC-nano-ESI-MS/MS analysis.

Protein identification by nanoUPLC-nano-ESI-MS/MS analysis
A total of 26/29 protein spots with significant differential Compared to motor branches of the femoral nerve, 13 protein spots exhibit significant differential expression in saphenous nerves, including seven up-regulated, four down-regulated, one peculiarly expressed and one with no expression.  expression were successfully detected using nanoUPLC-nano-ESI-MS/MS analysis, and all contained peptides with matching scores > 34 (P < 0.05). The peptide segment coverage rate was 1-64%. After excluding the most frequently observed contaminant peptides derived from keratin [10] and repetition, 12 proteins were differently expressed in sensory and motor nerve tissues ( Figure 3). Protein comparisons of motor branches of the femoral nerve revealed down-regulated expressions in saphenous nerve tissues of transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an anonymous protein product (gi|55628) were upregulated, and aldose reductase, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-like protein ( Table 1).
Comparisons of ventral roots revealed up-regulated expressions of lactoylglutathione lyase, adenylate kinase isozyme 1, two anonymous proteins products (gi|55628 and gi|1334163), and down-regulated expression of poly(rC)-binding protein 1 (PCBP1). Expression of rCG31027 was absent in dorsal root tissues ( Table 2). Figure 3 Peptide mass data of transgelin in two-dimensional gel electrophoresis map of saphenous nerves.
x-axis represents mass-to-charge ratio (m/z), whereas the y-axis represents relative intensity.
Peptide mass data were searched in the National Center for Biotechnology Information (NCBI) using the Mascot MS/MS Ions search engine and Rattus species category. The peptide sequence was KYDEELEER.
The protein spot was identified as transgelin, with protein sequence coverage of 64% (64% of peptide sequence is in accordance with the transgelin protein sequence).

DISCUSSION
With the characterization of sensitive, accurate, and specific quantitative methods, nanoUPLC-nano-ESI-MS/MS has been developed and widely used in fields of biology [11] , medicine [12] , pharmacology [13] , and food safety [14][15] . The present study employed proteomic techniques using nanoUPLC-nano-ESI-MS/MS to separately screen two groups of over-expressed proteins from proximal and distal segments of motor and sensory nerves. A total of 12 protein types were differentially expressed in sensory and motor nerve tissues, including several novel proteins specific to sensory or motor nerves. Three protein spots of transgelin, with significant differential expression in 2DGE maps, was expressed at higher levels in the saphenous nerve compared with the motor branch of the femoral nerve. Transgelin, also termed Sm22-alpha, is a transformation-sensitive and rapidly gelled actin that was initially discovered in chicken gizzard smooth muscle [16][17] , followed by smooth muscle cells in the aorta, uterus, lung, and intestine [18] . Transgelin expression is not restricted to smooth muscles, because the protein is also present in normal mesenchymal cells and secondary cultures of mouse and rat embryo fibroblasts, but is absent in many apparently normal fibroblast cell lines [19] . Transgelin down-regulation may, therefore, be an early and sensitive marker for the onset of transformation [19] . Transgelin, which often exhibits diminished expression in cancer, also regulates matrix metalloproteinases 9 expression, likely via interference with extracellular signal-regulated kinase 1 and 2 signaling [20] .
In the motor branch of the femoral nerve, expression of aldose reductase and a GAPDH-like protein was significantly increased compared to other proteins in the saphenous nerve. Aldose reductase is a monomeric 5,10-methylenetetrahydrofolate reductase-dependent oxidoreductase with wide substrate specificities for carbonyl compounds; it is generally used as the first step in fructose synthesis from glucose [21] . GAPDH catalyzes an important energy-yielding step in carbohydrate metabolism [22] -the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide. Although GAPDH has been commonly used as a housekeeping gene due to its stably and constitutively high level expression, researchers have reported varying regulation under specific conditions [23] . Increased expression of aldose reductase and GAPDH in motor branches could indicate that energy metabolism in motor nerves prefers the glycolysis and gluconeogenesis pathway, which takes place without ATP. A protein spot, which was expressed relatively greater in dorsal root 2DGE maps than in ventral roots, was identified as adenylate kinase isozyme 1. Adenylate kinase is a phosphotransferase enzyme that catalyzes reversible conversion of magnesium adenosine triphosphate + adenosine monophosphate to magnesium adenosine monophosphate + adenosine monophosphate, and also plays an important role in cellular energy homeostasis [24] . Adenylate kinase 1 is the cytosolic isozyme of adenylate kinase and is present in skeletal muscle, brain, and erythrocytes [25] . Up-regulation of this enzyme in sensory nerves could further reveal differences between energy metabolism pathways in motor and sensory nerves, although its exact role remains to be determined. In ventral root 2DGE maps, three protein spots were expressed greater than in dorsal roots and were identified as PCBP1. PCBP1 is a member of the heterogeneous ribonucleoprotein family and participates in transcription and translation regulation [26] . In the prespliceosomal complex, PCBP1 and small nuclear ribonucleoprotein U1 bind together and are associated with silencing of pseudoexon splicing [27] . In addition to serving as an important RNA-binding protein [28] , PCBP1 acts as a DNA-binding protein [29] . In a recent study, transcripts targeted by the lack of PCBP1 were identified, and some identified transcripts were grouped into neuronal categories. For example, microtubuleassociated protein tau, roundabout homolog 2, slit homolog 2, and semaphoring 6A were grouped into axonogenesis, neurogenesis, axon guidance, neurite morphogenesis, and neuron morphogenesis during differentiation [30] . Consistent with the present ventral root results, PCBP1 could serve as a specific protein marker for motor nerves.
It should be noted that although differentially expressed proteins in motor and sensory nerves were observed, consistency was low between proximal and distal nerve segments. Only an unnamed protein product (gi|1334163) was detected with high expression in the saphenous nerve and ventral roots. These results were likely due to different cells and extracellular matrix of proximal and distal nerve segments. In situ immunohistochemistry, as well as in vitro pure cell proteome analysis, could help to identify these mechanisms of action.
In conclusion, through the use of 2DGE and nanoUPLCnano-ESI-MS/MS analysis, comparisons between motor branches of the femoral nerve and saphenous nerve, as well as ventral and dorsal roots, several proteins were identified for the first time to be differentially expressed in sensory and motor nerves. These proteins could play roles in specific nerve regeneration. Based on these results, functional analyses of these differentially expressed proteins, as well as the differences between sensory and motor nerves following injury, should be performed in the further.

MATERIALS AND METHODS Design
A proteomics experiment.

Time and setting
The experiments were performed at the Institute of Orthopedics, Chinese PLA General Hospital and National Center of Biomedical Analysis, China from June 2010 to March 2011.

Materials
Nine female, specific pathogen-free, Wistar rats, weighing 250-280 g, were provided by the Experimental Animal Center of Academy of Military Science, China. The rats were housed at 26°C with 45-55% relative humidity, and were maintained in a 12-hour light/12-hour dark cycle. The rats were allowed free access to food and water.

Sensory and motor nerve harvesting
Following general phenobarbital anesthesia (40 mg/kg, intraperitoneal), the bilateral inguen was incised to expose the femoral nerve bifurcation. Motor branch segments to the quadriceps muscles and saphenous nerves, respectively, which were distal to the bifurcation, were carefully separated and transected using microinstruments. A midline skin incision was made in the lumbar region to expose lumbar vertebrae at the L 1-5 level, and the lamina were subsequently removed. Dura mater and arachnoid membranes were also incised, and segments of ventral and dorsal roots were gently and sequentially removed and carefully transected from the spinal cord using fine forceps.

Protein sample preparation
Total protein from two couples of sensory and motor nerve tissues were extracted according to a previously described method [31] . Briefly, nerve segments were homogenized in liquid nitrogen, transferred to an Eppendorf tube on ice, and further homogenized in buffer (40 mM Tris-base, 7 M urea, 2 M thiourea, 2% CHAPS, 1% dithiothreitol, and 1 mM ethylenediamine tetraacetic acid) at a ratio of 1: 10 (v/v). Subsequently, a 10-μL cocktail of protease inhibitors (0.7 mg/mL pepstatin A, 0.5 mg/mL leupeptin, 0.3 mg/mL EDTA-Na 2 , and 100 mg/mL phenylmethyl sulfonylfluoride; Sigma, St. Louis, MO, USA) was added to the Tris buffer. Following sonication, 5 μL of 10 mg/mL DNA and RNA enzyme (Sino-American Biotechnology, Montgomery, Pennsylvania, USA) were added to the sample. The homogenate was maintained on ice for 20 minutes, followed by centrifugation at 40 000 × g for 20 minutes at 4°C. The supernatant was collected and stored at -80°C. Protein was quantified using the Bradford method [32] .

2DGE and image analysis
A 2DGE protocol was performed as previously described [33] and was repeated three times. Briefly, 160 μg of nerve tissue proteins was loaded onto a ceramic immobilized pH gradient gel strip holder using the in-gel rehydration mode. The IPG gel was rehydrated for 12 hours under a low voltage of 30 V at 20°C. Isoelectric focusing was performed under the following parameters: 500 V for 1 hour, 1 000 V for 1 hour, and 8 000 V for 10 hours. Focused strips were placed in sodium dodecyl sulfate-balanced solution twice at room temperature for 15 minutes each, followed by 13% sodium dodecyl sulfate polyacrylamide gel vertical electrophoresis using the PROTEN II xi Cell system (Bio-Rad, Hercules, CA, USA) in a circulating bath at 16°C with 40 mA for 40 minutes and 60 mA for 5 hours, respectively. To ensure reliability, each type of nerve tissue was pooled and run on three separate gels. The gels were stained with silver using a mass spectrometry compatible method for clearer visualization [34][35] and were scanned using an image scanner. A total of three images from each nerve were analyzed using Imagemaster 2D platinum software (Amersham Pharmacia Biotech, Staffanstor, Sweden). Statistical comparisons (analysis sets using Student's t-test, P < 0.05) were performed between motor branches of femoral nerves and saphenous nerves, as well as ventral roots and dorsal roots. Protein spots with > 2-fold relative expression intensity differences and identical changes in repeated experiments were regarded as differential protein spots.

In-gel digestion
The stained protein spots were excised from the gel and digested using trypsin [36] . Briefly, after the gel pieces were rinsed three times with Milli-Q water, the samples were treated incubated in 50 mM NH 4 HCO 3 and 50% acetonitrile. The spots were then dehydrated for 10 minutes in 100% acetonitrile, dried at room temperature, and then rehydrated in a digestion buffer of 25 mM NH 4 HCO 3 containing 0.01 μg/μL modified trypsin. The gel slices were completely covered by digestion buffer and incubated overnight at 4°C. The tryptic peptides were extracted from the gel at room temperature, rinsed once in water, twice in 5% trifluoroacetic acid, and vortexed in 50% acetonitrile. The samples were finally concentrated by drying under vacuum conditions.