Talk:Bottom-up proteomics
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MagnusPalmblad (talk) 11:33, 24 April 2008 (UTC) Is "bottom-up proteomics" not used in a much wider sense than for the identification of spots from a 2D gel as presently stated in the article? Almost any method which measures peptides or their sequences (by MS/MS) to infer identity or quantity of proteins is described as "bottom-up" in the literature. This includes MudPIT and similar approaches. What is the difference between "bottom-up" and "shotgun" proteomics? If there isn't any, perhaps these pages should be merged? --MagnusPalmblad
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edit- I have seen shotgun presented as both a subset and separate from bottom-up; I'm not sure if there is a broad consensus. My suggestion would be to bring both this article and shotgun proteomics up to B class and see where we are. If you have some good references, you can put them in using the Pubmed ID and template filler.[1] Diagram(s) would be good, too. An overview should go in protein mass spectrometry and proteomics itself has been needing some work for a while. Much to do here. --Kkmurray (talk) 14:57, 24 April 2008 (UTC)
- How about something like this: "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. The proteins may first be purified by a method such as gel electrophoresis resulting in one or a few proteins in each proteolytic digest. Alternatively, the crude protein extract is digested directly, followed by one or more dimensions of separation of the peptides by liquid chromatography coupled to mass spectrometry, a technique known as shotgun proteomics. By comparing the masses of the proteolytic peptides or their tandem mass spectra with those predicted from a sequence database, peptides can be identified and multiple peptide identifications assembled into a protein identification." For the bibliography: Chait BT (2006). "Chemistry. Mass spectrometry: bottom-up or top-down?". Science (journal). 314 (5796): 65–6. doi:10.1126/science.1133987. PMID 17023639.
{{cite journal}}: Unknown parameter|month=ignored (help) and Aebersold R, Mann M (2003). "Mass spectrometry-based proteomics". Nature. 422 (6928): 198–207. doi:10.1038/nature01511. PMID 12634793.{{cite journal}}: Unknown parameter|month=ignored (help) From this it should be clear that shotgun proteomics is a subset of bottom-up proteomics. I'll see if I can make a diagram comparing bottom-up and top-down. --MagnusPalmblad (talk) 12:08, 25 April 2008 (UTC) - The analogy with DNA sequencing may be more confusing than enlightening. In de novo shotgun (or bottom-up) DNA sequencing, there is no template like a sequence database (the product of the sequencing). Instead short but overlapping sequences are assembled to longer "contigs", assembled into genes and chromosomes. Use of overlapping sequences from proteolytic peptides is hardly ever used in proteomics, as it is very difficult to get sufficient sequence coverage to assemble the full protein sequence. But as it is at least a theoretical possibility, using DNA sequencing as an analogy to bottom-up proteomics could be confusing. --MagnusPalmblad (talk) 12:29, 25 April 2008 (UTC)
- How about something like this: "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. The proteins may first be purified by a method such as gel electrophoresis resulting in one or a few proteins in each proteolytic digest. Alternatively, the crude protein extract is digested directly, followed by one or more dimensions of separation of the peptides by liquid chromatography coupled to mass spectrometry, a technique known as shotgun proteomics. By comparing the masses of the proteolytic peptides or their tandem mass spectra with those predicted from a sequence database, peptides can be identified and multiple peptide identifications assembled into a protein identification." For the bibliography: Chait BT (2006). "Chemistry. Mass spectrometry: bottom-up or top-down?". Science (journal). 314 (5796): 65–6. doi:10.1126/science.1133987. PMID 17023639.
- Here's another one that makes your point (if implicitly):VerBerkmoes NC, Bundy JL, Hauser L; et al. (2002). "Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis". J. Proteome Res. 1 (3): 239–52. PMID 12645901.
{{cite journal}}: Explicit use of et al. in:|author=(help)CS1 maint: multiple names: authors list (link). This is the earliest mention of bottom-up I could find: Kelleher, Neil L. (1999). ""Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry"". J. Am. Chem. Soc. 121 (4): 806–812. doi:10.1021/ja973655h.{{cite journal}}: Unknown parameter|coauthors=ignored (|author=suggested) (help). I'll try to track down a counter-example, but I think that you may be right that the general definition puts proteolysis approaches into the bottom-up category. --Kkmurray (talk) 14:52, 25 April 2008 (UTC)
- Here's another one that makes your point (if implicitly):VerBerkmoes NC, Bundy JL, Hauser L; et al. (2002). "Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis". J. Proteome Res. 1 (3): 239–52. PMID 12645901.
- I changed the entry according to the discussion above and added a figure. Please modify it if necessary (I am not a native speaker).--MagnusPalmblad (talk) 15:32, 28 April 2008 (UTC)
The key difference between top-down and bottom-up proteomics is the point at which data is collected in regard to protein digestion. In bottom-up proteomics enzymatic digestion (usually by trypsin) is done prior to any data collection and so there can be confusion as to the protein origin of isoforms and non-unique peptides. In top-down proteomics the data collection (eg quantitation, ID, PI and MW) is done on the intact purified protein. One could argue that digestion is required for ID, and that is true, but one could also say the same thing about collision cell fragmentation within the mass spectrometer. The point is that in top-down methodology protein purification is done prior to digestion/fragmentation which removes doubt about the origin of the fragments. For this reason 2D PAGE is an accepted top-down method.[1] I think both this page and the top-down page need editing to reflect this thinking.Stuart Christopher Brown (talk) 21:54, 15 June 2019 (UTC)
References
- ^ Proteomics 2014, 14, 872-889 https://doi.org/10.1002/pmic.201300424
Redundancy with other pages
editThis has been discussed a little bit on other topics, but seems reiterating: This page seems to be the same topic (differently stated/named differently), from these pages:
They should be merged for clarity? Photocyte (talk) 15:06, 1 May 2023 (UTC)
- Shotgun proteomics is probably the same thing as bottom-up proteomics and could be merged. But peptide mass fingerprinting is a distinct technique that doesn't involve tandem mass spectrometry. –CWenger (^ • @) 16:01, 1 May 2023 (UTC)
- The peptide mass fingerprinting page mentions MS/MS in several places (including the main figure), so the distinction may not be that clear. It seems to me that purely MS1 peptide mass fingerprinting is not done any more, and should be clearly stated as a historical technique and not bring MS/MS into it? Photocyte (talk) 03:54, 2 May 2023 (UTC)
- I think they are mentioning MS/MS as an alternative if peptide mass fingerprinting is insufficient for identification. It is confusing though, particularly the figure, which would benefit from the left (PMF) path being highlighted at least. –CWenger (^ • @) 14:39, 2 May 2023 (UTC)
- The peptide mass fingerprinting page mentions MS/MS in several places (including the main figure), so the distinction may not be that clear. It seems to me that purely MS1 peptide mass fingerprinting is not done any more, and should be clearly stated as a historical technique and not bring MS/MS into it? Photocyte (talk) 03:54, 2 May 2023 (UTC)