Abstract 4951: In-depth analysis of gene and protein expression levels in the colorectal adenocarcinoma cell line Caco-2
Authors: Slebos Robbert J. c. , Zhang Bing , Halvey Patrick J. , Liebler Daniel C. 
Published: February 2011

Abstract
Novel shotgun proteomic analysis platforms using liquid-chromatograpy tandem mass spectrometry (LC-MS/MS) allow in-depth surveys of thousands of proteins in a semi-quantitative manner. The colorectal carcinoma cell line Caco-2 was analyzed in triplicate by Affymetrix Hu-133 microarray and by multidimensional shotgun proteomic analysis using iso-electric focusing and analysis on an LTQ-Orbitrap instrument. MS-spectra were searched using Myrimatch against the NCBI database (version 36.51) in forward and reverse orientation. Proteins were included when identified by a minimum of 2 distinct peptides. The combined microarray and shotgun proteomics results were compared to a manual curation of protein immunohistochemistry (IHC) data from ProteinAtlas (www.proteinatlas.org). Microarray analysis yielded a total of 9861 expressed mRNAs which were observed in all 3 replicate analyses. Shotgun proteomic analysis identified 3896 protein groups (grouping eliminates redundancy from protein isoforms) while 2986 proteins were observed from ProteinAtlas. A comparison of microarray with shotgun proteome data showed that the proportion of proteins identified for each of 5 ranked mRNA expression categories increased with increasing mRNA levels. Of the top 20% most highly expressed mRNAs, 2600 (75%) were also identified by shotgun proteomics, while of the bottom 20% mRNAs only 384 (11%) could be identified by shotgun proteomics. When classified by GO categories for biological processes, molecular function or cellular location, the proportions of genes or proteins classified in each of the sub-categories did not differ substantially, suggesting that shotgun proteomics identifies a representative collection of proteins which is similar to mRNA distribution. A large majority of the proteins identified by shotgun analysis, 3695 (95%) of 3896, could be unambiguously mapped to genes from identified by microarray analysis. A much smaller overlap between mRNA and protein results was observed with ProteinAtlas data: only 2002 (67%) of 2986 proteins from ProteinAtlas were also found by microarray analysis. Only 37 proteins were found by both protein analysis methods and not by microarray, the large majority of which were immunoglobulins. In conclusion, our comparative analysis shows that shotgun proteomic analysis performed on a standardized platform provides an in-depth and unbiased protein expression profile, which largely follows the expression levels determined by gene expression analysis. Although it is unclear why almost 1,000 proteins identified by IHC are not detectable by either microarray or shotgun proteomic analysis, this apparent discrepancy may indicate a lack of antibody specificity. Future research will be needed to expand the coverage of shotgun proteomics to include all possible expressed proteins within a complex protein mixture.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4951.