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Updated: Jun 23, 2026
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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Proteomics by mass spectrometry: approaches, advances, and applications
John R Yates1, Cristian I Ruse, Aleksey Nakorchevsky
1Department of Chemical Physiology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA. jyates@scripps.edu
Annual Review of Biomedical Engineering
|April 30, 2009
Summary
Mass spectrometry (MS) is a powerful proteomics tool. This review details MS experimental design, instrumentation like the Orbitrap, and separation methods for quantitative phosphoproteomics.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Mass spectrometry (MS) is a key technology in large-scale proteomics.
- Understanding MS experimental design is crucial for effective proteomic analysis.
Purpose of the Study:
- To review the framework of MS experiments, including separation methods, instrumentation, and experimental design.
- To highlight the strengths and limitations of MS in proteomics.
- To provide guidance on selecting experimental designs for specific analytical goals.
Main Methods:
- Discussion of fundamental proteomic analyses and recent advancements.
- Emphasis on Orbitrap mass analyzer capabilities (high resolution, mass accuracy, dynamic range).
- Exploration of chromatographic techniques like multidimensional separation and ultra-performance liquid chromatography (UPLC).
- Detailed examination of Multidimensional Protein Identification Technology (MudPIT) for sample preparation, orthogonal separations, and data analysis.
- Application of MudPIT to quantitative phosphoproteomics, including enrichment, informatics, and simultaneous protein/phosphorylation level determination.
Main Results:
- The Orbitrap offers high resolution and mass accuracy, enhancing data acquisition and analysis.
- Multidimensional separation techniques, particularly MudPIT, are effective for complex proteomic samples.
- MudPIT enables quantitative phosphoproteomics, allowing for simultaneous measurement of protein expression and phosphorylation levels.
- Case study illustrates MudPIT application in quantitative phosphoproteomics of the beta adrenergic pathway.
Conclusions:
- Mass spectrometry, especially with advanced instrumentation like Orbitrap and techniques like MudPIT, is a versatile tool for large-scale proteomics.
- These methods facilitate in-depth analysis of biological systems, particularly in cell signaling proteomics.
- The review provides a framework for optimizing MS experimental design based on research objectives.
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