Peptides were extracted from gel pieces se quentially using 0. 4% formic acid in 3% ACN twice, once using 0. 4% formic acid in 50% ACN and once using 100% ACN. The extracted peptides were dried and stored at 80?C until LC MSMS analysis. In solution digestion Five hundred ug of depleted synovial fluid protein was recon stituted in 40 mM ammonium bicarbonate. It was then re duced, alkylated selleck chemical EPZ-5676 and digested overnight using trypsin as mentioned above. Strong cation exchange chromatography SCX was carried out as described earlier. Briefly, 200 ug of digested peptide mixture was acidified using 1 M phosphoric acid and equilibrated with 10 mM potas sium phosphate buffer containing 25% acetonitrile, pH 2. 85 and fractionated using SCX Inhibitors,Modulators,Libraries on a Poly sulfoethyl A column using an Agilent 1200 HPLC system containing a binary pump, UV detector and a fraction collector.
The peptides were eluted using a salt gradient be tween solvent A and solvent B. Twenty six fractions obtained from the fraction ation were completely dried, reconstituted in 0. 1% trifluor oacetic acid, and further desalted using stage tips packed with C18 material. Desalted fractions were dried in speedvac and reconstituted in 10 ul of 0. 1% TFA prior Inhibitors,Modulators,Libraries to reversed phase liquid chromatography based tandem mass spectrometry analysis. OFFGEL fractionation Approximately 300 ug of in solution digested depleted tryptic peptides was used for isoelectric point based frac tionation using Agilents 3100 OFFGEL fractionator. As per the manufacturers protocol, peptides were separated using pH 3 10 IPG strip.
The peptides were focused for 50kVh with maximum current of 50 uA and maximum voltage set to 4000 V. Twelve fractions were collected after fractionation and then acidified using 1% TFA prior to sample cleaning using stage tips. Lectin affinity enrichment Approximately 10 mg of the total protein pooled from five OA samples was Inhibitors,Modulators,Libraries diluted in 10 mM phosphate buffer, pH Inhibitors,Modulators,Libraries 7. 8. For glycoprotein enrichment, the samples were incubated with a mixture of three agarose conjugated lectins concanavalin A, wheat germ agglutinin and jacalin for 12 h at 4?C. The beads were then washed three times using wash buffer and the bound pro teins were eluted using a mixture of carbohydrates. The eluate was dialyzed to Inhibitors,Modulators,Libraries remove free sugars and then concentrated using 3 kDa cut off filters. The protein concentration was estimated by Lowrys method.
Two hundred and fifty selleck ug of the enriched protein frac tion was then resolved by SDS PAGE. Twenty six gel bands were excised and subjected to in gel trypsin diges tion procedure as described in the previous section. Two hundred and fifty ug of the enriched glycoprotein was also subjected to SCX fractionation as described earlier. Twenty fractions were collected and desalted using stage tips as mentioned above.