arch engines. Restrictions were placed on species, mass tolerance, maximum missed cleavages by trypsin and cysteine modification by carbamidomethylation. Statistics For comparison of group differences concerning anthropometric data and pain intensity the Kruskal Wallis test was applied using IBM SPSS; p,0.05 was considered significant. In-gel digestion by trypsin In the gel used for protein identification 300 mg proteins were loaded and analyzed as 11805219 above. Protein spots were excised using a homemade spot picker. The picked protein spots were digested with trypsin. Briefly, the gel pieces were washed with a mixture of acetonitrile/ ammonium bicarbonate, dehydrated with acetonitrile and incubated with 30 ml of 20 mg/ml trypsin overnight at 37uC. Silver stained protein spots 
were destained with 15 mM potassium ferricyanide/50 mM sodium thiosulfate as described previously before trypsination. The supernatant was transferred to a new tube and the peptides further extracted from the gel by incubation in 50% acetonitrile/5% trifluoroacetic acid for about 3 hours at room temperature during constant mixing. The supernatant obtained by the two steps pooled, dried by SpeedVac. Results Protein concentration The total protein concentrations were measured before 2-DE analysis; 74 mg/ml in the TM pool, 55 mg/ml in the CWP pool and 59 mg/ml in the CON pool. The samples were desalted, concentrated and 50 mg protein from each group could be analyzed by 2-DE. 2-DE analysis About 300 protein spots could be detected. 98 protein spots that were of good quality for identification were picked and in-gel digested for identification by nLC- MS/MS and MALDI-TOF mass spectrometer. It was possible to identify 18690793 97 of the protein spots. The apparent molecular weight and isoelectric point determined from 2-DE pattern were generally in agreement with the theoretical values with the identified proteins. 50% of the proteins had different identity according to the accession numbers, i.e., many of the identified proteins were expressed as different isoforms. That could be explained by post translational modification and truncation of the proteins. The majority of identified proteins are known muscle proteins pertaining to several functional classes, i.e., metabolic, structural, regulatory, and contractile proteins and proteins that are involved in inflammatory responses. Identified proteins known to be involved in nociceptive and pain processes were alpha-1 antitrypsin, creatine kinase, nerve grow factor, carbonic anhydrase, myoglobin, fatty acid binding protein and actin aortic smooth muscle. Protein identification by LC-MS/MS The dried Oleandrin web tryptic samples from fluorescently stained proteins were dissolved in 6 ml of 0.1% formic acid. Peptides were analyzed using an on-line nano-flow HPLC system in conjugation with the mass spectrometer HCTultra PTM Discovery System. A 100 mm675 mm C18 column was used for separation at a flow rate 300 nL/min. The gradient buffers used were 0.1% formic acid in water and 0.1% formic acid in acetonitrile and a linear gradient from 0100% buffer B in 40 min was used for separation. The automated online tandem MS analysis was performed using collision induced dissociation of peptide ions. Protein identification by MALDI-TOF The dried tryptic samples from silver stained proteins were dissolved in 4 ml of 0.1% trifluroacetic acid. The peptides were mixed 1:1 with matrix solutions consisting of dihydroxybenzoic acid in 70% ACN/0.3% TFA, and 1 ml was the
