Editing Proteomics (section)

====Mass spectrometry and protein profiling====
{{main|Mass spectrometry}}
[[File:Thermo - Finnigan LCQ Mass Spectrometer (15797493459).jpg|thumb|LCQ Mass Spectrometer used in mass spectrometry.]]
There are two mass spectrometry-based methods currently used for [[Proteomic profiling|protein profiling]]. The more established and widespread method uses high resolution, two-dimensional electrophoresis to separate proteins from different samples in parallel, followed by selection and staining of differentially expressed proteins to be identified by mass spectrometry. Despite the advances in 2-DE and its maturity, it has its limits as well.
The central concern is the inability to resolve all the proteins within a sample, given their dramatic range in expression level and differing properties. The combination of pore size, and protein charge, size and shape can greatly determine migration rate which leads to other complications.<ref name="Weston & Hood 2004">{{cite journal | vauthors = Weston AD, Hood L | title = Systems biology, proteomics, and the future of health care: toward predictive, preventative, and personalized medicine | journal = Journal of Proteome Research | volume = 3 | issue = 2 | pages = 179–196 | year = 2004 | pmid = 15113093 | doi = 10.1021/pr0499693 | citeseerx = 10.1.1.603.4384 }}</ref>

The second quantitative approach uses stable isotope tags to differentially label proteins from two different complex mixtures.<ref>{{Citation |last1=Rozanova |first1=Svitlana |title=Quantitative Mass Spectrometry-Based Proteomics: An Overview |date=2021 |work=Quantitative Methods in Proteomics |volume=2228 |pages=85–116 |editor-last=Marcus |editor-first=Katrin |place=New York, NY |publisher=Springer US |language=en |doi=10.1007/978-1-0716-1024-4_8 |isbn=978-1-0716-1023-7 |last2=Barkovits |first2=Katalin |last3=Nikolov |first3=Miroslav |last4=Schmidt |first4=Carla |last5=Urlaub |first5=Henning |last6=Marcus |first6=Katrin |series=Methods in Molecular Biology |pmid=33950486 |s2cid=233740602 |editor2-last=Eisenacher |editor2-first=Martin |editor3-last=Sitek |editor3-first=Barbara|doi-access=free }}</ref><ref>{{Citation |last1=Nikolov |first1=Miroslav |title=Quantitative Mass Spectrometry-Based Proteomics: An Overview |date=2012 |url=https://link.springer.com/10.1007/978-1-61779-885-6_7 |work=Quantitative Methods in Proteomics |volume=893 |pages=85–100 |editor-last=Marcus |editor-first=Katrin |access-date=2023-04-14 |place=Totowa, NJ |publisher=Humana Press |language=en |doi=10.1007/978-1-61779-885-6_7 |isbn=978-1-61779-884-9 |last2=Schmidt |first2=Carla |last3=Urlaub |first3=Henning|series=Methods in Molecular Biology |pmid=22665296 |hdl=11858/00-001M-0000-000F-C327-D |s2cid=33009117 |hdl-access=free }}</ref> Here, the proteins within a complex mixture are labeled isotopically first, and then digested to yield labeled peptides. The labeled mixtures are then combined, the peptides separated by multidimensional liquid chromatography and analyzed by tandem mass spectrometry. Isotope coded affinity tag (ICAT) reagents are the widely used isotope tags. In this method, the cysteine residues of proteins get covalently attached to the ICAT reagent, thereby reducing the complexity of the mixtures omitting the non-cysteine residues.

[[Quantitative proteomics]] using stable isotopic tagging is an increasingly useful tool in modern development. Firstly, chemical reactions have been used to introduce tags into specific sites or proteins for the purpose of probing specific protein functionalities. The isolation of phosphorylated peptides has been achieved using isotopic labeling and selective chemistries to capture the fraction of protein among the complex mixture. Secondly, the ICAT technology was used to differentiate between partially purified or purified macromolecular complexes such as large RNA polymerase II pre-initiation complex and the proteins complexed with yeast transcription factor. Thirdly, ICAT labeling was recently combined with chromatin isolation to identify and quantify chromatin-associated proteins. Finally ICAT reagents are useful for [[proteomic profiling]] of cellular organelles and specific cellular fractions.<ref name="Weston & Hood 2004"/>

Another quantitative approach is the accurate mass and time (AMT) tag approach developed by [[Richard D. Smith]] and coworkers at [[Pacific Northwest National Laboratory]]. In this approach, increased throughput and sensitivity is achieved by avoiding the need for tandem mass spectrometry, and making use of precisely determined separation time information and highly accurate mass determinations for peptide and protein identifications.