Journal of Proteome Research the APCNull phenotype. Several novel pathways are implicated in the current proteomic data set including DNA topoisomerase activity, terpenoid backbone biosynthesis and α6-β4 integrin signaling. Functional enrichment results from transcriptomic data alone (data not shown) showed considerable divergence from those of proteomics data. Known APCNull phenotypic pro- perties were either not observed (e.g., negative regulation of the cell cycle) or showed only modest enrichment in transcriptomic only analyses (e.g., actin-binding, adjusted p-value = 0.371; cell cell adherens junction, adjusted p-value = 0.378). Loss of CDH1 expression, a previously validated hallmark of APCNull cells, is weakly corroborated at the transcript level (log2 ratio = 0.2, adjusted p-value = 0.45) but clearly demonstrated at the proteo- mic level (log2 ratio = 1, adjusted p-value = 0.1). Furthermore, target enrichment of the EMT-related ZEB1 transcriptional repressor in SW480APC is only observed in the proteomics data set. Therefore, proteomics appears to present a more reliable approach to biological discovery in the SW480APC model and underscores the added value of proteomic techniques over tran- scriptomic approaches. Label free shotgun proteomics provides signiﬁcant depth of proteome coverage and aids in the detection of subtle proteomic diﬀerences in the SW480APC model. Nonetheless, there are some important limitations associated with the approach. The proteomic consequences of APC restoration were only examined in a single SW480APC clone, raising the possibility that clonal cell line features contribute to observed proteomic diﬀerences. However, Faux and colleagues showed that multiple SW480APC clones display a consistent set of phenotypic properties, for ex- ample, decreased growth rates, loss of anchorage independent growth, increased CDH1 levels.11 Our proteomic data recapitu- late many of these features. In particular, we document decreased expression of over 20 cell adhesion proteins (e.g., DSP, CTNND1, CADM1) and changes to cell cycle regulatory pro- teins (e.g., RB1, SMARCA4) in SW480Null. LC MS/MS and LC MRM proteomic data for CDH1 (2- and 3-fold higher in SW480APC, respectively) agree with the previously reported 2-fold decrease.11 These speciﬁc ﬁndings suggest that proteomic diﬀerences primarily reﬂect the biological consequences of APC restoration. As with most proteomic technologies, there is a bias toward detection of high abundant proteins and many lower abundant proteins are either not detected at all, or do not produce enough signal to allow a conﬁdent comparison of relative expression levels between samples. In the SW480APC model several proteins from the Wnt signaling pathway (e.g., APC, AXIN2, TCF7, LEF1, DVL1) were not detected. Consequently, only a partial evaluation of the proteomic eﬀects of APC restoration on the Wnt signaling pathway can be made. Likewise many transcription factors, which may play key regulatory roles in the SW480APC model, are not detected. However, the high number proteins conﬁdently identiﬁed in this data set (>5000) still allows many signiﬁcant diﬀerences to be observed and meaningful biological information may be inferred from these diﬀerences. For instance, enhanced activity of the transcriptional repressor ZEB1 in SW480Null can be deduced from elevated levels of ZEB1 protein targets in SW480APC. Furthermore, veriﬁcation of 22 proteins by targeted LC MRM conﬁrmed the direction and magnitude of the diﬀerences observed in the LC MS/MS proteomics data, thereby demonstrating the general reliability of the approach. The approach used here to analyze the eﬀects restoration of wildtype APC can be applied to other model systems where known ARTICLE phenotypic diﬀerences arise from a speciﬁc genetic alteration. In colorectal cancer mutations in TP53, KRAS and SMAD4 contribute signiﬁcantly to tumor development.5 Elucidation of the global pro- teomic consequences of these mutations should yield new insights into basic tumor biology. In conclusion, we have demonstrated the utility of label free shotgun proteomics to assess the proteomic eﬀects of a single gene diﬀerence in a colorectal cancer cell model. Our ﬁndings reﬂect previously reported phenotypic features of the APCNull phenotype while also identifying new aspects of the underlying biology associated with APC deﬁciency. ’ ASSOCIATED CONTENT bS Supporting Information Supplemental tables. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *Robbert J. C. Slebos, Ph.D., Vanderbilt University School of Medicine, Medical Research Building III, U1213, 465 21st Avenue South, Nashville, TN 37232. Tel: 615-936-3063. Fax: 615-936-1001. E-mail: r.slebos@vanderbilt.edu. ’ ACKNOWLEDGMENT For purposes of clarity and brevity, proteins have been abbreviated to HGNC gene symbols. This work was supported in part by NIH grants U54CA126479 (to D.C.L.) and R01GM088822 (to B.Z.). We thank Misti Martinez, Kristin Cheek, and Sarah Stuart for technical assistance. We thank Zhiao Shi for programming assistance. ’ REFERENCES (1) Ahrens, C. 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