View source for Physics:Bottom-up proteomics

[[Image:Top-down_vs_bottom-up_proteomics_image.tif|right|350px|thumb|Top-down vs bottom-up proteomics]]
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'''Bottom-up proteomics''' is a common method to identify proteins and characterize their amino acid sequences and [[Biology:Post-translational modification|post-translational modification]]s by [[Biology:Proteolysis|proteolytic digestion]] of proteins prior to analysis by [[Physics:Mass spectrometry|mass spectrometry]].<ref name="pmid12634793">{{cite journal |vauthors=Aebersold R, Mann M |title=Mass spectrometry-based proteomics |journal=Nature |volume=422 |issue=6928 |pages=198–207 |date=March 2003 |pmid=12634793 |doi=10.1038/nature01511 |bibcode=2003Natur.422..198A }}</ref><ref name="pmid17023639">{{cite journal |author=Chait BT |title=Chemistry. Mass spectrometry: bottom-up or top-down? |journal=Science |volume=314 |issue=5796 |pages=65–6 |year=2006 |pmid=17023639 |doi=10.1126/science.1133987}}</ref> 
The major alternative workflow used in [[Biology:Proteomics|proteomics]] is called [[Physics:Top-down proteomics|top-down proteomics]] where intact proteins are purified prior to digestion and/or fragmentation either within the mass spectrometer or by 2D electrophoresis.<ref>{{cite journal |vauthors=Wright EP, Partridge MA, Padula MP, Gauci VJ, Malladi CS, Coorsen JR |title=Top-down proteomics: Enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection |journal=Proteomics |year=2014 |volume=14 |issue=7–8 |pages=872–889 |doi=10.1002/pmic.201300424 |pmid=24452924 |doi-access=free }}</ref> 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.<ref>{{cite web|title=Bottom-up Proteomics|url=http://planetorbitrap.com/bottom-up-proteomics#.WhOjfNjJCUk|website=PlanetOrbitrap|publisher=Thermo Fisher Scientific|access-date=20 November 2017}}</ref> 

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 [[Physics:Shotgun proteomics|shotgun proteomics]].<ref>{{cite journal |vauthors=Washburn MP, Wolters D, Yates JR |title=Large-scale analysis of the yeast proteome by multidimensional protein identification technology |journal=Nat. Biotechnol. |volume=19 |issue=3 |pages=242–247 |year=2001 |pmid=11231557 |doi=10.1038/85686 }}</ref><ref>{{cite journal |vauthors=Wolters DA, Washburn MP, Yates JR |title=An automated multidimensional protein identification technology for shotgun proteomics |journal=Anal. Chem. |volume=73 |issue=23 |pages=5683–5690 |year=2001 |pmid=11774908 |doi=10.1021/ac010617e }}</ref> By comparing the masses of the proteolytic peptides or their [[Physics:Tandem mass spectrometry|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.<ref name=yates>{{cite journal |vauthors=Yates JR, Ruse CI, Nakorchevsky A |title=Proteomics by Mass Spectrometry: Approaches, Advances, and Applications |journal=Annu. Rev. Biomed. Eng. |year=2009 |volume=11 |pages=49–79 |doi=10.1146/annurev-bioeng-061008-124934 |url=http://download.bioon.com.cn/view/upload/month_0910/20091014_179880bfc9f5eeb2548cnpX5P835Y2Cb.attach.pdf |pmid=19400705}}</ref>

==Disadvantages==
There is limited protein sequence coverage by identified peptides, loss of labile PTMs, and ambiguity of the origin for redundant
peptide sequences.<ref name=yates/> 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.<ref>{{cite journal|last1=Zhang|first1=Yaoyang|last2=Fonslow|first2=Bryan R.|last3=Shan|first3=Bing|last4=Baek|first4=Moon-Chang|last5=Yates|first5=John R. |title=Protein analysis by shotgun/bottom-up proteomics|journal=Chem Rev|date=2014-04-10|volume=113|issue=4|page=2343<!--|pages=2394-->|doi=10.1021/cr3003533|pmid=23438204|pmc=3751594}}</ref>

==See also==
*[[Biology:Protein mass spectrometry|Protein mass spectrometry]]
*[[Physics:Shotgun proteomics|Shotgun proteomics]]
*[[Peptide mass fingerprinting]]
*[[Physics:Top-down proteomics|Top-down proteomics]]

==References==
{{Reflist}}

[[Category:Mass spectrometry]]
[[Category:Proteomics]]



{{Sourceattribution|Bottom-up proteomics}}