HGNC gene symbol ADD3 ASS1 CADM1 CDH1 CLU CST1 CTNND1 DKK4 DSG2 DYSF EGFR FDPS LCP1 LGALS3BP LLGL2 MGAT1 NES PDCD4 PPL PYCR1 VIL1 VSNL1 39 63 25 39 40 40 85 100 41 124 26 41 21 59 18 14 112 48 173 101 82 75 40 30 5 18 12 7 47 2 19 8 70 19 23 22 1 2 258 12 106 37 26 30 0.1 1 2.2 1 1.6 2.4 0.7 5.5 1 3.8 1.5 1 0.2 1.3 4.1 2.7 1.3 1.9 0.6 1.3 1.6 1.2 QL p-value AdjP 5.15 9.10 1.75 3.44 1.70 3.83 8.30 1.64 6.49 2.54 3.10 3.83 3.80 1.59 3.79 2.98 9.44 1.69 4.09 5.15 5.09 1.57 10 1 10 6 10 3 10 3 10 5 10 5 10 5 10 6 10 4 10 9 10 4 10 4 10 1 10 4 10 4 10 3 10 5 10 4 10 3 10 4 10 6 10 3 8.36 4.04 7.21 1.06 5.67 8.00 1.39 1.37 3.77 6.37 2.38 2.78 7.67 1.63 2.78 9.84 1.41 1.66 1.16 3.47 2.55 6.67 10 1 10 3 10 2 10 1 10 3 10 3 10 2 10 3 10 2 10 6 10 2 10 2 10 1 10 2 10 2 10 2 10 2 10 2 10 1 10 2 10 3 10 2 LC MRM log2 (SW480Null/ SW480APC) 0.3 0.5 1.3 1.7 2.1 1.8 1.2 6 1.2 0.9 1.6 1 0.2 1.5 1.4 1 1.2 2.3 1.2 1.9 1.5 1.1 ARTICLE t test, p-value 10 3 10 1 10 4 10 4 10 4 10 4 10 4 10 4 10 3 10 2 10 2 10 4 10 2 10 3 10 2 10 2 10 2 10 4 10 4 10 3 10 2 10 2 9.80 1.37 1.00 1.00 1.00 6.00 1.00 1.00 1.80 1.14 3.05 2.00 9.33 9.60 4.00 2.21 4.57 5.00 2.00 1.60 1.53 1.77 Journal of Proteome Research Table 2. Summary of LC MS/MS and LC MRM-MS Data for 22 Proteins Validated by Targeted Proteomics SW480APC count SW480Null count LC MS/MS log2 (SW480Null/ SW480APC) adhesion and motility, thereby contributing to a more tumoro- genic phenotype APCNull cells. Proteins up-regulated in SW480Null cells included cell cycle- related proteins RB1, SMARCA4, CENPF and DNA topoisomerases TOP2A, TOP2B (Figure 3A). Among other proteomic diﬀer- ences observed, SW480Null cells display up-regulated EGFR, but down-regulated NGFR, which suggests a shift in growth factor response. SW480Null cells display down-regulation of SLC5A1 (sodium glucose cotransporter) and SLC12A6 (potassium chlor- ide cotransporter). Down-regulation of MVD and FDPS, two key regulators of terpenoid backbone synthesis synthesis, may aﬀect the prenylation of signaling enzymes. These changes may be the result of increased growth rates in SW480Null cells. To broaden the search for enriched functional protein groups, we carried out a limited manual inspection of signiﬁcantly diﬀeren- tially expressed proteins. This approach reveals functional groups which fall outside the Webgestalt enrichment criteria (i.e., those groups not in the top ﬁve ranked categories based on adjusted p-values), as well as proteins which share a common function but lack a common GO designation. Manual interrogation (g3 proteins, all up in SW- 480Null) revealed elevated levels of proteins involved in mRNA processing (DDX23, RBM10, SART1, CD2BP2, SFRS2IP) and nuclear transport (NUP50, NUP155, XPO4). Assessment of the impact of these protein expression changes would require further experiments. However, the magnitude of these protein changes is similar to those associated with well-documented eﬀects on cell adhesion, cytoskeletal organization and cell cycle regulation. Thus, these newly identiﬁed proteomic changes provide new avenues to explore the APCNull phenotype. Verification of Proteomic Differences by LC MRM-MS A subset of the proteins found to be diﬀerentially expressed by LC MS/MS shotgun proteomics were assessed by LC MRM-MS. This approach provides a quantitative measurement of proteo- typic peptides and an accurate assessment of the relative pro- tein abundance in SW480APC and SW480Null.25 LC MRM-MS measurements showed substantial reproducibility across three replicate runs. For 22 proteotypic peptides analyzed, a median CV value of 11.5% was observed. Correlation between log ratios (SW480APC/SW480Null) from each approach was also evalu- ated (r = 0.63, Spearman ranked correlation). For all proteins, LC MRM-MS measurements agree with LC MS/MS shotgun proteomics, and identical trends in protein expression were observed between the two platforms, that is, proteins with high levels in SW480APC by shotgun proteomics are also seen to be high by LC MRM-MS (e.g., DKK4, CDH1) (Figure 4 and Table 2). Similarly, proteins which were not found to be diﬀerentially expressed by shotgun proteomics, showed no diﬀerence by LC MRM-MS (e.g., LCP1, ADD3). Thus, the data demonstrate that for a subset of diﬀerentially expressed proteins, label free shotgun proteomics data and LC-MRM data are broadly concordant. ’ DISCUSSION Extensive information is currently available on genomic varia- tion in cancer. Large data repositories such as the Cancer Genome Atlas (TCGA) and Catalogue of Somatic Mutations in Cancer (COSMIC) provide vast knowledge on a range of mutations across a spectrum of tumor types.2,6 However, the global proteomic eﬀects 1191 dx.doi.org/10.1021/pr2009109 |J. Proteome Res. 2012, 11, 1184–1195