Analytical Characteristics of Cleavable Isotope-Coded Affinity Tag-LC-Tandem Mass Spectrometry for Quantitative Proteomic Studies  Cecily P. Vaughn, David K. Crockett, Megan S. Lim, Kojo S.J. Elenitoba-Johnson  The Journal of Molecular Diagnostics  Volume 8, Issue 4, Pages 513-520 (September 2006) DOI: 10.2353/jmoldx.2006.060036 Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic of an ICAT-labeling experimental protocol comparing control and test samples. Protein from each sample is collected, denatured, and reduced. Equal quantities of each sample are then labeled with the ICAT reagents. Typically, the control sample is labeled with the light reagent, and the test sample is labeled with the heavy reagent. The samples are then combined and digested with trypsin. Digested samples are fractionated using a cation exchange column. Each fraction is run through an avidin affinity column to separate ICAT-labeled peptides from unlabeled peptides. The flow-through sample, containing unlabeled peptides, is collected from the avidin affinity column first, followed by elution of the ICAT-labeled peptides. The flow-through sample may be retained for analysis by LC-MS/MS. The ICAT-labeled sample is subjected to LC-MS/MS analysis and quantification. The Journal of Molecular Diagnostics 2006 8, 513-520DOI: (10.2353/jmoldx.2006.060036) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 The number of peptide and protein identifications versus the number of fractions analyzed. Multiple fractions were collected for four of the ICAT experiments. The number of peptides and proteins identified in these experiments is shown relative to the number of fractions analyzed. As expected, increasing the number of fractions, and therefore reducing the relative complexity of each fraction analyzed, generally results in identification of more peptides and proteins. The Journal of Molecular Diagnostics 2006 8, 513-520DOI: (10.2353/jmoldx.2006.060036) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Percentages of peptides with cysteine residues and with quantification data. The percentages of peptides containing cysteine residues (light gray bars) and the percentages of peptides with quantification data (dark gray bars) for each of six separate ICAT experiments is shown. Although only 24.4 to 40.8% of the peptides contained cysteine residues, the majority of the cysteine-containing peptides had quantification data. The Journal of Molecular Diagnostics 2006 8, 513-520DOI: (10.2353/jmoldx.2006.060036) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Percentages of proteins quantified and differentially expressed. The percentages of proteins with quantification data in each of six separate ICAT experiments are shown. Between 15.9 and 36.4% (mean, 27.0%) of the total number of proteins identified had quantification data (light gray bars). Proteins differentially expressed by 1.5-fold or greater (dark gray bars) comprised between 6.1 and 21.1% (mean, 14.3%) of the total identified proteins. The Journal of Molecular Diagnostics 2006 8, 513-520DOI: (10.2353/jmoldx.2006.060036) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Classification of proteins by subcellular localization. The percentages and average percentages, respectively, of proteins for each cellular location are shown for proteins from the non-cICAT-labeled sample (light gray bars) and the quantified proteins from the cICAT experiments (dark gray bars). The percentages in each category are comparable between the unlabeled proteins and the quantified proteins. The Journal of Molecular Diagnostics 2006 8, 513-520DOI: (10.2353/jmoldx.2006.060036) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Classification of proteins by molecular function. The percentages and average percentages, respectively, of proteins for each molecular function are shown for proteins from the non-cICAT-labeled sample (light gray bars) and the quantified proteins from the cICAT experiments (dark gray bars). The percentages in each category are comparable between the unlabeled proteins and the quantified proteins. The Journal of Molecular Diagnostics 2006 8, 513-520DOI: (10.2353/jmoldx.2006.060036) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 7 Western blot validation of protein expression. The control and test protein samples from experiment 4 were resolved on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was probed with an anti-actin antibody as a loading control. The membrane was also probed with antibodies against two proteins identified by cICAT-LC-MS/MS, α-tubulin, and PLK-1. The expression levels shown by Western blot are concordant in trend with the cICAT ratio for each protein. The Journal of Molecular Diagnostics 2006 8, 513-520DOI: (10.2353/jmoldx.2006.060036) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions