Supplementary information to G02201A- Isolation of peptides from CAD by tryptic
“in gel” digestion and Zip Tips
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.
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.