The Study of Biological Systems through Proteomics: A Quantitative Proteomic Analysis of Non-small Cell Lung Cancer (NSCLC)

摘要

In the post-genomic era proteomics has emerged as one of the most important areas of research, particularly with respect to differential proteomics, the comparative analysis of the normal versus disease states of distinct proteomes. The most well established tool for proteomic analysis is 2-dimensional gel electrophoresis (2-DE). This tool offers a simple method to visually identify changes in protein abundance. 2-DE has been a staple in the examination of proteomes, and has undergone numerous technological advances over the past several decades. Isobaric tags for relative and absolute quantitation (iTRAQ) is a non-gel based alternative to 2-DE as a tool for proteomic analysis. Over the last decade or more it has become a common alternative to 2-DE technology coupled with mass spectrometry. The global use and evolution of proteomics warrants incorporation of the recent advances and uses of the technology into the undergraduate laboratory curriculum to prepare students for the proteomic era. In data presented herein we utilize iTRAQ coupled with mass spectrometry as a novel approach to teach students about the identification, quantification, statistical analysis and road blocks associated with the ever advancing field of comparative proteomics. Introduction From the time when the first prokaryotic and eukaryotic genomes were released in 1995 and 1996 respectively [1,2], the field of genomics has exploded into the 21st century providing an enormous amount of information, from the complete sequencing of the human genome to global gene expression in cells, uncovering a vast, fertile landscape for bioinformatics exploration.However, despite the success of these efforts, coupling gene sequence with function remains an ongoing effort.Predictably, however, another discipline, proteomics, has been expanding alongside these technologies.Proteomics describes the methodical identification and quantification of proteins expressed in biological systems.Application of proteomics allows a researcher to obtain information on protein identity, expression level, variants, and post-translational modifications.Researchers attempted to get a head start on this type of analysis twenty years before the first genome sequences were presented [3,4].It all began with the introduction of 2-dimensional electrophoresis (2-DE) and the separation of complex mixtures for comparative expression patterns.This provided much excitement for insightful interactions in human health, agriculture, environment and biotechnology would be uncovered. 2-DE has been the most favored technology for decades, to investigate the global qualitative and quantitative proteomic changes of proteomes [5-9].2-DE gel electrophoresis offers a low cost method of protein analysis. However, it has several notable shortcomings, particularly with respect to variations in repetitive gel runs, ultimately making protein analysis a very labor-intensive and experimentally error-prone task [10]. Although modifications in methodology have helped to alleviate such issues [11] making it a reliable technology for protein profiling.Despite the global use of 2-DE in proteomic research, there is a lack of proteomics education represented in the undergraduate laboratory curriculum to prepare the future workforce.The Rochester Institute of Technology is one of the few colleges that has successfully incorporated its introduction into the science curriculum [12].There are most likely several reasons but cost, reproducibility and implementation within a semester course seem to be the most likely barriers for more widespread acceptance into undergraduate science curricula. With advances in liquid chromatography (LC), mass spectrometry (MS) and bioinformatics, newer comparative and quantitative studies are possible.LC-MS/MS is a technique coupling high pressure liquid chromatography and tandem mass spectrometry to identify protein mixtures [13, 14].When coupled with chemical tagging, LC-MS/MS allows for quantifi