<p>Supplementary information to G02201A- Isolation of peptides from CAD by tryptic “in gel” digestion and Zip Tips</p><p>Purified CAD protein was phosphorylated by active MAPK as described in the accompanying manuscript, Graves et al. (1). The phosphorylated CAD protein was separated on 7.5 % SDS-PAGE and the phosphorylated protein excised and digested “in gel” with trypsin exactly as described by the method of Leonard and Hell (2). Sequencing grade typsin was obtained from Promega (Madison, WI). The trypsin digest was performed for 24 hr at 37 0C and the peptides extracted from the gel with 200 l 50 mM ammonium bicarbonate (pH 8.0) and 200 l 50% acetonitrile twice. The recovery of radioactive peptides was typically 90%. The extracted peptides were concentrated in a Speed-Vac, acidified with trifluoroacetic acid (TFA) and applied to Zip Tips (Millipore, Bedford, MA) equilibrated with 0.1% TFA according to the manufacturers procedure. The Zip Tips were washed with 0.1% TFA , then water and the peptides eluted in step fractions of 2, 5, 10, 20 and 50% acetonitrile/water. These fractions were analyzed for radioactivity and the 10 and 20 % acetonitrile fractions selected for further analysis by mass spectrometry.</p><p>Analysis of CAD peptides by Nanoelectrospray mass spectrometry The Zip Tip fractions were loaded into Gold-coated borosilicate capillaries drawn to a tip diameter of one micron. The tips were used as emitters for a commercial off-axis nanoElectrospray source coupled to a triple quadrupole mass spectrometer of QhQ geometry (Micromass, Beverly, MA.). The sampling cone potential was set at 34V, extractor at 5V and the spray capillary was set to 940V. Source block temperature was maintained at 120C and the resolution was typically set to 300 or higher. CID experiments were performed with ultrapure Ar at a target thickness of 30 x 1014 atom/cm2. Neutral loss spectra were acquired at 35eV by scanning the first and final quadrupoles while maintaining a constant offset in their scan cycles.1 MS/MS scans for consecutive loss of water and phosphate (optimized from studies of the synthetic peptide, VYFLPIpTPHYVTQVIR) identify a doubly-charged peptide in the digest mixture consistent with the expected mass of residues 450-465 incorporating a single phosphorylation site. References 1. Graves, L.M., Guy, H.I., Kozlowski, P., Huang, M. Lazarowski, E., Pope, R.M., Collins, M.A., Earp, H.S. and Evans, D.R. Nature (in press). 2. Leonard, A.S. and Hell, J.W., J. Biol. Chem. 272, 12107-12115, 1997. 3. Jaffee, H., Veeranna, Shetty, K.T. and Pant, H.C., Biochemistry, 37, 3931-3940, 1998.</p>