Shotgun Proteomics for Analysis of Cancer‐Relevant Tissue Proteotypes |
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| Launch in standalone player | |
| Air date: | Friday, February 06, 2009, 10:00:00 AM |
| Category: | Proteomics |
| Description: | Shotgun proteomics using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) provides the most powerful analytical platform for global inventory of complex proteomes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. These features of shogun proteomics data pose challenges in comparing proteomes from different biological states. We have implemented a standardized shotgun proteomics analytical platform based on isoelectric focusing of peptide mixtures, followed by reverse phase LC‐MS/MS on Thermo LTQ and LTQ‐Orbitrap instruments. Here we describe the development of a statistical approach for comparison of spectral count data from shotgun proteomic datasets and we apply this method to compare proteomes cancer‐relevant tissue specimens and cell lines. These comparisons yield proteotypes, which comprise distinguishing proteomic characteristics of phenotypes. To this end, we have developed statistical and visualization strategies that reveal the biological differences between tissue specimens. We have compared proteomes of normal colon tissue, colon adenomas and adenocarcinomas, as well as proteomes from a collection of colon cancer cell lines that differ in expression of DNA mismatch repair genes. We demonstrate that these shotgun proteomic datasets are sufficiently rich in quantitative information that they can be analyzed for statistically significant differences and that these global comparisons of proteomes yield meaningful biological information. We also have compared shotgun proteomic analyses of frozen and formalin‐fixed, paraffin‐embedded (FFPE) specimens prepared from the same colon adenoma tissues. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C‐terminal to arginine C‐terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, crosslinked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to two days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5 and 10 years. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery. http://proteome.nih.gov |
| Author: | Daniel Liebler, Ph.D. |
| Runtime: | 75 minutes |
| CIT File ID: | 14900 |
| CIT Live ID: | 7344 |
| Permanent link: | http://videocast.nih.gov/launch.asp?14900 |