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An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics
Abstract
We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.
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Citations
Cited by
Mass spectrometry-based proteomics
Aebersold, R., & Mann, M. (2003) · Nature · doi:10.1038/nature01511 · Cited by 6,955
Comparing protein abundance and mRNA expression levels on a genomic scale
Greenbaum, D. (2003) · Genome biology · doi:10.1186/gb-2003-4-9-117 · Cited by 1,692
Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., & Gygi, S. P. (2003) · Journal of Proteome Research · doi:10.1021/pr025556v · Cited by 1,647
Protein Analysis by Shotgun/Bottom-up Proteomics
Zhang, Y., Fonslow, B. R., Shan, B., Baek, M.-C., & Yates, J. R. (2013) · Chemical Reviews · doi:10.1021/cr3003533 · Cited by 1,491
Self-citationProteomics by Mass Spectrometry: Approaches, Advances, and Applications
Yates, J. R., Ruse, C. I., & Nakorchevsky, A. (2009) · Annual Review of Biomedical Engineering · doi:10.1146/annurev-bioeng-061008-124934 · Cited by 1,078
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Metadata
- Authors
- Wolters, D. A., Washburn, M. P., & Yates, J. R.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2001-10-25
- Volume
- 73
- Issue
- 23
- Pages
- 5683-5690
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- OpenAlex
- W1970696723
- Cited by
- 1,819
- References
- 0
- FWCI
- 45.34
- Type
- journal article
- Language
- en
- Open access
- closed
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