ernatant was taken and dialyzed to 50 mM Tris, pH 8.0. For rough cytosolic protein isolation, 400 ml buffer was added per ml of ground cell material. The sample was then centrifuged at 20 000 g for 40 min at 4 C. The supernatant fraction was used directly for the isolation of phosphopeptides. Isolation of phosphopeptides from nuclear extracts by IMAC Approximately 40 mg of nuclear extract were reduced with 2 mg of DTT, alkylated with 10 mg of iodoacetamide, and digested AZ-6102 chemical information overnight at 37 C with 4 mg of trypsin. The digest was terminated by the addition of formic acid to a final concentration of 3%. To desalt the sample, it was loaded onto Oligo R3, which was filled into constricted gel loader tips. The bound peptides were washed with 0.1 M acetic acid and eluted with 0.1 M HAc containing 50% acetonitrile. The eluate was diluted to 30% acetonitrile and incubated in batch with $5 ml of POROS MC material, which was charged with FeCl3. After 30 min of incubation at room temperature the suspension was filled into a constricted gel loader tip. The bound peptides were washed with 0.1 M HAc containing 30% acetonitrile and eluted with 1 M NH4OH containing 30% acetonitrile. IMAC protocol used for isolation of phosphopeptides from cytosolic extracts Proteins were precipitated from 300 mg of cytosolic extracts as described previously. The protein precipitate was dissolved in 8 M urea in 0.5 M NH4HCO3 and reduced and alkylated as described above. The solution was diluted to 0.8 M urea and proteins were digested with 5 mg of trypsin as described above. To desalt the sample, 40 ml of settled Oligo R3 was filled into constricted 200 ml tips. The sample was loaded and bound peptides were washed with 0.1% HAc and eluted with 70% acetonitrile in 0.1% HAc. Thereafter, the sample was dried in a speed vac. To reduce nonspecific binding of acidic peptides to the IMAC column, peptide carboxyl groups were esterified in methanolic HCl PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19816862 as described previously. Methylesterified peptides were dried in a speed vac and redissolved in a solution containing equal parts of methanol, acetonitrile and 0.02% HAc as described previously. The peptide solution was Nucleic Acids Research, 2006, Vol. 34, No. 11 3269 incubated with 5 
ml of IMAC material. After 1 h of incubation the suspension was filled into a constricted gel loader tip, washed with 0.02% HAc and bound peptides were eluted with 125 mM of Na2HPO4. Analysis of phosphopeptides by nano-LC-MS/MS and MS/MS/MS IMAC eluates were separated on a nano-reversed phase highperformance liquid chromatography . Samples were applied to a trapping column using 0.1% TFA at a flow rate of 20 ml/ min. Bound peptides were eluted to a 75 mm 150 mm analytical column of the same material at a flow rate of 250 nl/min. Elution was performed by applying a linear gradient of 2.540% ACN in 0.1% formic acid in 3 h. The HPLC was coupled online to an LTQ linear ion trap mass spectrometer, which was equipped with a nanoelectrospray ion source. Distal coated silica nanospray capillaries of New Objective were used and the electrospray voltage was set to 1500 V. The mass spectrometer was operated in the data-dependent mode: 1 full scan was followed by MS/MS scans of the four most abundant ions. These ions were excluded from further selection for 30 s. For each MS2 spectrum the neutral loss algorithm in the Xcalibur 1.4 software was enabled. In this mode, MS/MS/MS experiments are automatically triggered, if a neutral loss of phosphoric acid is detected