Bottom-Up Proteomics
From Handwiki File:Top-down vs bottom-up proteomics image.tif 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
- Protein mass spectrometry
- Shotgun proteomics
- Top-down proteomics
References
- ↑ "Mass spectrometry-based proteomics". Nature 422 (6928): 198–207. March 2003. doi:10.1038/nature01511. PMID 12634793. Bibcode: 2003Natur.422..198A.
- ↑ Chait BT (2006). "Chemistry. Mass spectrometry: bottom-up or top-down?". Science 314 (5796): 65–6. doi:10.1126/science.1133987. PMID 17023639.
- ↑ "Top-down proteomics: Enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection". Proteomics 14 (7–8): 872–889. 2014. doi:10.1002/pmic.201300424. PMID 24452924.
- ↑ "Bottom-up Proteomics". Thermo Fisher Scientific. http://planetorbitrap.com/bottom-up-proteomics#.WhOjfNjJCUk.
- ↑ "Large-scale analysis of the yeast proteome by multidimensional protein identification technology". Nat. Biotechnol. 19 (3): 242–247. 2001. doi:10.1038/85686. PMID 11231557.
- ↑ "An automated multidimensional protein identification technology for shotgun proteomics". Anal. Chem. 73 (23): 5683–5690. 2001. doi:10.1021/ac010617e. PMID 11774908.
- ↑ 7.0 7.1 "Proteomics by Mass Spectrometry: Approaches, Advances, and Applications". Annu. Rev. Biomed. Eng. 11: 49–79. 2009. doi:10.1146/annurev-bioeng-061008-124934. PMID 19400705. http://download.bioon.com.cn/view/upload/month_0910/20091014_179880bfc9f5eeb2548cnpX5P835Y2Cb.attach.pdf.
- ↑ 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. PMID 23438204.
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Categories: [Mass spectrometry] [Proteomics]
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