In-Gel Tryptic Digestion and Peptide Characterization

Protein bands obtained from the binding assay were subjected to in-gel digestion with trypsin^{80 (link),81 (link)}. The digested peptide solution was desalted and concentrated, and was eluted using a homemade C18 nano-column (100–300 nl with trypsin of POROS reverse-phase R2 material (20–30 μm in bead size, PerSeptive Biosystems, Foster City, CA)) and 1.5 ul of 50% MeOH, 49% H₂O, and 1% HCO₂H. qTOF-MS of the eluted peptides was performed using a Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer (AB Sciex Instruments, Framingham, MA) equipped with an electrospray ionization (ESI) source. The quadrupole analyzer was used to select precursor ions for fragmentation in the hexapole collision cell. The produced ions were analyzed using an orthogonal TOF analyzer and fitted with a reflector, a micro-channel plate detector, and a time-to-digital converter.