Proteomic Analyses using High-Efficiency Separations and Accurate Mass Measurements
While the development of analytical techniques for detecting and identifying proteins has been an actively pursued area of research for many years, a recent and concerted reemergence of work in this area has resulted in an explosion of new developments that can be applied across multiple disciplines. The advent of numerous mass spectrometric and separation advances may be viewed as the technical driving force behind these developments, but other factors are perhaps equally crucial. These factors include the increasing availability of sequences for many organisms as a result of recent advancements in genome analysis, recognition that there are limitations to using sequence/transcriptome information alone, and the more apparent value of directly characterizing the biomolecules and pathways that actually drive cellular events. Direct identification of structural and functional proteins allows perturbations at the protein level to be linked to responses at the cellular level, and provides a method for analyzing global proteomic changes in virtually any model system. The field of proteomics, in particular, has benefited from numerous recent analytical advances that have provided increased sensitivity and dynamic range of proteins detected, as well as analysis throughput. Traditional approaches typically have involved separations using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) in conjunction with a mass spectrometric (MS) component for protein identification.[1] These seminal techniques set the foundation for the advancing field of proteomics. The limitations associated with the gel-based techniques are well known and stem mostly from a lack of sufficient dynamic range needed for in-depth proteome coverage and [2] and from the large amount of time needed for analysis, which dramatically limits throughput. Significant developments have been made in an attempt to address both these and other issues. With the advent of electrospray ionization, researchers can now couple multi-dimensional liquid chromatography (LC) separations with MS analysis. Combined with a ?bottom up? strategy, in which proteins are digested into smaller peptide fragments for identification, the LC-MS platform has proved highly successful for identifying peptides (and generally proteins) from complex samples. This chapter discusses proteome analysis techniques that are based on high resolution LC separations and high mass accuracy MS instrumentation, specifically the use of Fourier transform ion cyclotron resonance MS (FTICR), and their application for high throughput global identification of proteins. The simultaneously expanded sensitivity, dynamic range, and mass accuracy afforded by FTICR place this system at the forefront of analytical systems that can attain proteomic identifications from complex biological samples. We will begin the discussions by describing a high throughput proteomics approach that capitalizes on the attributes of FTICR. The critically important supporting components that combine to provide a comprehensive proteomic analysis capability?electrospray ionization efficiency, pre-MS high efficiency LC separations, and downstream data processing methods?will then be discussed with the intent of providing insight into the process as a whole, as well as to highlight analytical challenges that remain to be addressed. Because quantitative measurements are so important to understanding even the simplest biological systems, some of the quantitation techniques and approaches being developed and applied in our laboratory are also discussed. Several applications are exemplified prior to concluding this chapter.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 891435
- Report Number(s):
- PNNL-SA-43219; TRN: US200621%%892
- Resource Relation:
- Related Information: Separations in Proteomics , 363-385
- Country of Publication:
- United States
- Language:
- English
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