Editing Proteomics (section)

===High-throughput proteomic technologies===
Proteomics has steadily gained momentum over the past decade with the evolution of several approaches. Few of these are new, and others build on traditional methods. Mass spectrometry-based methods, affinity proteomics, and micro arrays are the most common technologies for large-scale study of proteins.

====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.

==== Affinity proteomics ====
Affinity proteomics uses antibodies or other affinity reagents (such as oligonucleotide-based aptamers) as protein-specific detection probes.<ref>{{cite journal | vauthors = Stoevesandt O, Taussig MJ | title = Affinity proteomics: the role of specific binding reagents in human proteome analysis | journal = Expert Review of Proteomics | volume = 9 | issue = 4 | pages = 401–414 | date = August 2012 | pmid = 22967077 | doi = 10.1586/epr.12.34 | s2cid = 19727645 }}</ref> Currently this method can interrogate several thousand proteins, typically from biofluids such as plasma, serum or cerebrospinal fluid (CSF). A key differentiator for this technology is the ability to analyze hundreds or thousands of samples in a reasonable timeframe (a matter of days or weeks); mass spectrometry-based methods are not scalable to this level of sample throughput for proteomics analyses.

====Protein chips====
Balancing the use of mass spectrometers in proteomics and in medicine is the use of protein micro arrays. The aim behind protein micro arrays is to print thousands of protein detecting features for the interrogation of biological samples. Antibody arrays are an example in which a host of different antibodies are arrayed to detect their respective antigens from a sample of human blood. Another approach is the arraying of multiple protein types for the study of properties like protein-DNA, protein-protein and protein-ligand interactions. Ideally, the functional proteomic arrays would contain the entire complement of the proteins of a given organism. The first version of such arrays consisted of 5000 purified proteins from yeast deposited onto glass microscopic slides. Despite the success of first chip, it was a greater challenge for protein arrays to be implemented. Proteins are inherently much more difficult to work with than DNA. They have a broad dynamic range, are less stable than DNA and their structure is difficult to preserve on glass slides, though they are essential for most assays. The global ICAT technology has striking advantages over protein chip technologies.<ref name="Weston & Hood 2004"/>

====Reverse-phased protein microarrays====
[[File:Mechanism-of-AHA-bonding-to-Amino-Acids.svg|thumb|312x312px|Mechanisms showing how AHA labels onto proteins and where biotin-FLAG-alkyne tags mark the amino acid. Hand Drawn via Sigma Aldrich]]
This is a promising and newer microarray application for the diagnosis, study and treatment of complex diseases such as cancer. The technology merges [[Laser capture microdissection|laser capture microdissection (LCM)]] with micro array technology, to produce reverse-phase protein microarrays. In this type of microarrays, the whole collection of protein themselves are immobilized with the intent of capturing various stages of disease within an individual patient. When used with LCM, reverse phase arrays can monitor the fluctuating state of proteome among different cell population within a small area of human tissue. This is useful for profiling the status of cellular signaling molecules, among a cross-section of tissue that includes both normal and cancerous cells. This approach is useful in monitoring the status of key factors in normal prostate epithelium and invasive prostate cancer tissues.  LCM then dissects these tissue and protein lysates were arrayed onto nitrocellulose slides, which were probed with specific antibodies. This method can track all kinds of molecular events and can compare diseased and healthy tissues within the same patient enabling the development of treatment strategies and diagnosis. The ability to acquire proteomics snapshots of neighboring cell populations, using reverse-phase microarrays in conjunction with LCM has a number of applications beyond the study of tumors. The approach can provide insights into normal physiology and pathology of all the tissues and is invaluable for characterizing developmental processes and anomalies.<ref name="Weston & Hood 2004"/>