Bottom-up proteomics

Top-down vs bottom-up proteomics

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] BUP techniques can be an alternative to Maldi-Tof MS approaches, as they allow the identification of bacterial strains and the characterization of potential resistance and virulence factors in a single run.[3] 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.[4] 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.[5]

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.[6][7] 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

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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.[8]

## Disadvantages

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There is limited protein sequence coverage by identified peptides, loss of labile PTMs, and ambiguity of the origin for redundant peptide sequences.[8] 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.[9]

## See also

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* Protein mass spectrometry
* Shotgun proteomics
* Peptide mass fingerprinting
* Top-down proteomics

## References

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1. ^ 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.
2. ^ Chait BT (2006). "Chemistry. Mass spectrometry: bottom-up or top-down?". Science. 314 (5796): 65–6\. doi:10.1126/science.1133987. PMID 17023639.
3. ^ Present and future perspectives on mass spectrometry for clinical microbiology, Megan S. Gant, Julia Chamot-Rooke, Article 105296
4. ^ 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 24452924.7–8&rft.pages=872-889&rft.date=2014&rft_id=info:doi/10.1002/pmic.201300424&rft_id=info:pmid/24452924&rft.aulast=Wright&rft.aufirst=EP&rft.au=Partridge,+MA&rft.au=Padula,+MP&rft.au=Gauci,+VJ&rft.au=Malladi,+CS&rft.au=Coorsen,+JR&rft_id=https://doi.org/10.1002%2Fpmic.201300424&rfr_id=info:sid/en.wikipedia.org:Bottom-up+proteomics" class="Z3988">
5. ^ "Bottom-up Proteomics". PlanetOrbitrap. Thermo Fisher Scientific. Retrieved 20 November 2017.
6. ^ 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.
7. ^ 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.
8. ^ 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.
9. ^ 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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