Aliquots of each digest (1 ll) were transferred to a freshlypolished MALDI plate and overlayed with freshly-prepared matrix containing 4 mg/ml a-cyano-4-hydroxycinnamic acid, 50% CH3CN 0.1% formic acid (v/v), 0.1% TFA (v/v), and 5 mM(NH4)Cl. Data were acquired utilizing an Applied Biosystems 4800 MALDI TOF/TOF (Framingham, MA) mass spectrometer. An MS scan for the m/z range of 800–4000, averaging data from 1200 laser shots, was acquired in reflector positive ion mode. The 4000 Series explorer was then used to generate a list of at most 10 peaks above a minimum signal-to-noise ratio for subsequent MS/MS analysis. MS/MS scans were the average of 2000 laser shots acquired (1 kV, positiveion mode).