Parallel processing. Eiwei Amounts Topotecan of protein digestion and peptide fractionation of opportunities contr 17-DMAG and the treated cells were mixed and for digestion by the method of the filter-based sample preparation. Briefly, the lysate in a buffer containing SDS was solubilized on Microcon YM 30 aircraft and SDS was loaded from the samples followed by urea exchange removed by alkylation with 50 mM iodoacetamide. Urea was then replaced with 20 mM ammonium bicarbonate, before the proteins Were digested overnight at 37 with trypsin. The peptides were from the filter by centrifugation through elution with add Tzlichem collected water followed. 70 repetitive of each biological peptide mixture was separated into six fractions by strong anion exchange chromatography as described. Briefly, peptides developed on the S Pillars, made of 200 l micropipette tip with six layers of a data carrier Loaded support 3 M Empore anion exchange stacked. We used Britton and Robinson universal buffer of 20 mM acetic Acid, 20 mM phosphorus Acid, 20 mM boric composed Acid and with NaOH to the desired pH for Equilibration and elution fractions. The peptides were loaded at pH 11 and the fractions are then with buffer Eluted solutions of pH 8, 6, 5, 4 and 3. The eluted fractions were loaded onto peptide reverse phase C18 StageTips. The peptides were washed twice with buffer B containing 20 l 80% acetonitrile in 0.5% acetic Organic acid and L Solvents were eluted eluted in a SpeedVac concentrator prior to analysis of the mixture of peptides from LC-MS. Sequential enrichment with TiO2 phosphopeptides enrichment phosphopeptide beads, the peptides were eluted with trifluoroacetic Acid anges Acidified at pH 2.7 and ACN was added to a final concentration of 30%. After measuring the UV absorption, the samples were incubated with peptide beads TiO2 peptide of 1:4 or beads is 4:1.
At successive incubations, the peptide was bead suspension incubated for 30 min and centrifuged and the supernatant was incubated with a further aliquot Fra YEARS Prepared Riger TiO2 beads n for enriching Chsten. To avoid unspecific binding, the beads were again solution of TiO2 in a 30 mg / ml L Suspended prepared in 75% S Dihydrobenzoic acid ACN and 0.1% TFA. After incubation with the peptide mixture, the beads were washed with 30% ACN and 3% TFA twice followed by two washes with 75% ACN and 0.3% TFA. The phosphopeptides were then eluted with an elution buffer with ammonium hydroxide 15% and 40% ACN. Closing Lich eluted phosphopeptides were loaded on C18 StageTips. For LC MS / MS phosphopeptides were eluted as described above for the peptides, au He that the elution buffer of 60% ACN in acetic acid At 0.5% was described. Mass spectrometry of peptide Hordenine mixtures were measured with a HPLC system nanoflow nLC coupled conveniently via a nanoelectrospray ion source to a mass spectrometer LTQ Orbitrap Velos. Peptide chromatographic separation was in a 20 cm-S Column, which was in the house with Pur C18 AQ Reprosil 1.8m resin packed in buffer A. The peptides were made with a tile Rate of 200 nl / min with a linear gradient of 5% to 80% buffer B over 120 min or 200 depending on the experiment eluted. At the end of the gradient of the S Column was washed with 90% buffer B and with a buffer of 5%.