Gary A. Valaskovic and Neil L. Kelleher Pages 1 - 12 ( 12 )
Off-line miniaturized “nano-spray” formats for electrospray ionization mass spectrometry (ESI-MS) enable the routine identification of femtomole quantities of protein or peptide. Even greater strides have been achieved using on-line miniaturized ESI-MS methods, such as nanobore LC-MS and CE-MS. On-line methods enable greater sensitivity (sub-attomole limit of detection), dynamic range, and throughput. In either off- or on-line methods for protein analysis, samples are typically isolated and digested enzymatically, with MS analysis of the peptide fragments, yielding 5-50 percent sequence coverage, in a “bottom-up” approach. Obtaining biologically relevant (structure / function) information (such as the localization of regions of error or post-transnational modifications) often demands 100 percent sequence coverage and this may be obtained by analyzing intact proteins by MS with a “top-down” methodology. Proteome wide success with top-down methods will require the development of novel miniaturized approaches for sample preparation along with new tools for bioinformatics. As these miniaturized formats continue to power proteomics applications, they will undoubtedly pollinate “cross-over” applications in LC-MS ranging from drug discovery to development. An example of metabolite identification using an order of magnitude less sample than usually required, with a concurrent order of magnitude increase in signal, illustrates the potential of miniaturized formats in lead characterization activities.
Mass Spectrometry-Based Proteomics, electrospray ionization mass, spectrometry (ESI-MS), Proteome, LC-MS, electrospray ionization (ESI), matrix-assisted laser-desorption ionization (MALDI), PEPTIDE-DRIVEN PROTEOMICS, ICAT Isotope-Coded Affinity
New Objective, Inc., Woburn,MA; Dept. of Chemistry, University of Illinois at Urbana-Champaign, USA