Titansphere tio2 5 μm

For sample preparation, 5mg of the Gn-HCL protein lysate were first reduced with 5 mM TCEP and further alkylated with 50 mM iodoacetamide. Alkylated proteins were further diluted using 50mM Ammonium Bicarbonate to bring final Gn-HCL concentration to 0.6M and then digested with trypsin (1:50, trypsin: lysate ratio, Promega) for 16 h at 37°C. The overnight digests were clarified with brief spin and the supernatant pH was adjusted around pH2 using 10% TFA. Sep-Pak (Waters) columns were washed with methanol and washing buffer (2%acetonitrile/0.1%TFA) followed by loading of total peptides. The column was washed 3 times with washing buffer (2%acetonitrile/0.1%TFA) and final peptide elution was performed using high acetonitrile containing Elution buffers (50% and 80%acetonitrile/0.1%TFA). The peptide mixture was dried using speed vac. The dried peptide pellet was dissolved using Phthalic acid Buffer (0.1% P.Acid/20% Water/80% Acetonitrile, 2.5%TFA) and mixed with TiO2 (Titansphere 5 μm, GL sciences) beads and mixed on a rotator for two hours. The beads were washed two times with Phthalic acid Buffer followed by washing with 80% acetonitrile/0.1%TFA and finally 0.1% TFA. Phosphopeptides were eluted using 0.3M NH₄OH and the pH was adjusted around 2 using 50% TFA. The Phosphopeptides were dried using speed vac and further clarified using C18 mini columns as per above mentioned buffers.