bottom-up proteomics

Image: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.{{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 }}{{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}}

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. [https://www.sciencedirect.com/journal/microbes-and-infection/vol/26/issue/7 Present and future perspectives on mass spectrometry for clinical microbiology], Megan S. Gant, Julia Chamot-Rooke, Article 105296

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.{{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 }} 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.{{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}}

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.{{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 }}{{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 }} 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.