Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. [1] [2] The major alternative workflow used in proteomics is called top-down proteomics where intact proteins are purified prior to digestion and/or fragmentation either within the mass spectrometer or by 2D electrophoresis. [3] Essentially, bottom-up proteomics is a relatively simple and reliable means of determining the protein make-up of a given sample of cells, tissues, etc. [4]
In bottom-up proteomics, the crude protein extract is enzymatically digested, followed by one or more dimensions of separation of the peptides by liquid chromatography coupled to mass spectrometry, a technique known as shotgun proteomics. [5] [6] By comparing the masses of the proteolytic peptides or their tandem mass spectra with those predicted from a sequence database or annotated peptide spectral in a peptide spectral library, peptides can be identified and multiple peptide identifications assembled into a protein identification.
Advantages
For high throughput bottom-up methods, there is better front-end separation of peptides compared with proteins and higher sensitivity than the (non-gel) top-down methods. [7]
Disadvantages
There is limited protein sequence coverage by identified peptides, loss of labile PTMs, and ambiguity of the origin for redundant peptide sequences. [7] Recently the combination of bottom-up and top-down proteomics, so called middle-down proteomics, is receiving a lot of attention as this approach not only can be applied to the analysis of large protein fragments but also avoids redundant peptide sequences. [8]
See also
References
- ^ Aebersold R, Mann M (March 2003). "Mass spectrometry-based proteomics". Nature. 422 (6928): 198–207. Bibcode: 2003Natur.422..198A. doi: 10.1038/nature01511. PMID 12634793.
- ^ Chait BT (2006). "Chemistry. Mass spectrometry: bottom-up or top-down?". Science. 314 (5796): 65–6. doi: 10.1126/science.1133987. PMID 17023639.
- ^ Wright EP, Partridge MA, Padula MP, Gauci VJ, Malladi CS, Coorsen JR (2014). "Top-down proteomics: Enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection". Proteomics. 14 (7–8): 872–889. doi: 10.1002/pmic.201300424. PMID 24452924.
- ^ "Bottom-up Proteomics". PlanetOrbitrap. Thermo Fisher Scientific. Retrieved 20 November 2017.
- ^ Washburn MP, Wolters D, Yates JR (2001). "Large-scale analysis of the yeast proteome by multidimensional protein identification technology". Nat. Biotechnol. 19 (3): 242–247. doi: 10.1038/85686. PMID 11231557.
- ^ Wolters DA, Washburn MP, Yates JR (2001). "An automated multidimensional protein identification technology for shotgun proteomics". Anal. Chem. 73 (23): 5683–5690. doi: 10.1021/ac010617e. PMID 11774908.
- ^ a b Yates JR, Ruse CI, Nakorchevsky A (2009). "Proteomics by Mass Spectrometry: Approaches, Advances, and Applications" (PDF). Annu. Rev. Biomed. Eng. 11: 49–79. doi: 10.1146/annurev-bioeng-061008-124934. PMID 19400705.
- ^ Zhang, Yaoyang; Fonslow, Bryan R.; Shan, Bing; Baek, Moon-Chang; Yates, John R. (2014-04-10). "Protein analysis by shotgun/bottom-up proteomics". Chem Rev. 113 (4): 2343. doi: 10.1021/cr3003533. PMC 3751594. PMID 23438204.
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