Number of proteins identified in each ICAT experimenta

Proteins identifiedConfidence level (%)No. of proteins in conditioned mediumNo. of proteins in membrane preparations
Expt 1aExpt 1bExpt 2Expt 3Expt 1Expt 2
MMPI/vehicle ICAT ratio ≤0.77≥9910812243963747
MMPI/vehicle ICAT ratio ≥1.50≥99131622241
  • a Shown is the number of proteins identified from ICAT-labeled tryptic peptides from conditioned medium or membrane preparations of MMP-14-transfected MDA-MB-231 breast cancer cells in each experiment at confidence levels assigned by Mascot of ≥99% and ≥95%. Full lists of proteins and peptides are given in Tables S1 and S2 in the supplemental material. The ICAT ratio cutoff for decreased ICAT ratios of ≤0.77 was based upon ratios obtained for known substrates (see text and Table 2 for details). For high ratios, a cutoff of ≥1.5 was chosen, as previously defined (129). Experiments 1a and 1b are technical replicates in which the same ICAT-labeled conditioned medium sample was subjected to different strong cation-exchange separation (1a, an elution gradient of 0 to 500 mM salt; 1b, an elution gradient of 0 to 250 mM salt) prior to mass spectrometry (see Table S3 in the supplemental material).