[65] with some minor modifications: The A-1155463 ic50 protein spots were excised from Coomassie stained gels loaded with 100 μg protein. A piece of gel without staining was used as a negative control. The gel pieces were cut into approx. 1 mm3 pieces and washed twice for 15 min., first with water and second with water/acetonitrile 1:1 (v/v). The gel particles were then washed in acetonitrile to dehydrate

the gel (they shrunk and became white). A volume of 10 mM dithiotreitol (DTT) in 100 mM NH4HCO3 to cover the gel pieces was added and the proteins were reduced for 45 min at 56°C. After cooling, the DTT solution was replaced by the same volume of 55 mM iodoacetamide in 100 mM NH4HCO3 and the reduced proteins were alkylated for 30 min. in the dark. The gel pieces were then washed with water,

water/acetonitrile 1:1 (v/v) and acetonitrile to dehydrate the gel. Ice-cold digestion buffer containing 12.5 ng/μl trypsin in 50 mM NH4HCO3 was added to the gel pieces in a volume just buy Barasertib sufficient to rehydrate the gel (5-10 μl). After 45 min incubation on ice bath the unabsorbed digestion buffer was removed and replaced by 20 μl of 50 mM NH4HCO3 buffer to cover the gel pieces. The click here proteins were digested overnight at 37°C. The buffer solution with protein digest was recovered and kept at -20°C. Micropurification of peptides and loading on MALDI target The peptide solutions were purified on nano-scale reversed-phase columns prior to mass spectrometric analysis by the method described by Gobom et al [66]. The columns were prepared by loading a few μl slurry of a reversed phase chromatographic medium (Poros R2 10 μm, Applied Biosystems) dissolved in acetonitrile into a partially constricted GelLoader pipette tip. The column was packed by applying pressure with a syringe giving a column height of 4-10 mm and equilibrated with 1% TFA. The peptide digest was loaded onto the column and desalted by washing with 1% TFA. The peptides were eluted with matrix solution containing 5 μg/μl α-cyano-4-hydroxycinnamic acid in 70% acetonitrile and 0.1% TFA directly in one droplet onto the MALDI target

(Opti-TOF® 384 Well MALDI Plate Inserts, Tacrolimus (FK506) Applied Biosystems, California, USA). MALDI TOF/TOF tandem MS MALDI peptide mass spectra and MS/MS spectra of selected peptides were obtained on a 4800 Plus MALDI TOF/TOF™ Analyzer (Applied Biosystems). External mass calibration was done using a tryptic digest of beta-lactoglobolin (m/z 837.48 and 2313.26) and in some cases peaks from trypsin auto-digestion peptides (m/z 842.51 and 2211.12) were used for internal calibration of the peptide mass spectra. MS and MS/MS mass spectra were obtained at a laser intensity of 3000 and 3600 respectively. Peak lists were generated with an in house macro (in the Protein Research Group at Department of Biochemistry and Molecular Biology, University of Southern Denmark) using Data Explorer (Applied Biosystems) and converted to .mgf files containing the combined data from MS and MS/MS spectra for a sample.