{"id":13,"date":"2003-01-29T14:38:34","date_gmt":"2003-01-29T18:38:34","guid":{"rendered":"https:\/\/www.bumc.bu.edu\/ftms\/modern-instrumental-biochemistry\/proteomics-introduction\/"},"modified":"2012-07-29T00:33:45","modified_gmt":"2012-07-29T04:33:45","slug":"proteomics-introduction","status":"publish","type":"page","link":"https:\/\/www.bumc.bu.edu\/ftms\/modern-instrumental-biochemistry\/proteomics-introduction\/","title":{"rendered":"Proteomics Introduction"},"content":{"rendered":"
Introduction to Proteomics<\/em><\/div>\n

Proteomics, broadly speaking, is the study of proteins, usually in the context of identity, quantity, and structure of the protein and its modifications in a cell or tissue as a function of time or some external change. The most common method for generating maps of proteins in a cell at a particular time is the use of 2D-Gel electrophoresis.\"\"<\/p>\n

2D-Gel electrophoresis separates proteins by hydrodynamic volume in the vertical axis and by pH in the horizontal axis and is able to spread out many proteins from a batch of cells at the same time. However, there are many problems with 2D-Gels. First, they are notoriously difficult to reproduce consistently. Second, they are confined to hydrophilic proteins in a pH band from ~4-~10 so they will not detect any proteins that are highly acidic, highly basic, or hydrophobic. Third, they can detect proteins over a limited concentration range so that the best dynamic range possible is ~100x. Finally, they require many cells to generate these plots and a staining procedure to see the peaks.<\/p>\n

Mass spectrometry<\/a> in combination with various types of liquid chromatography is able to solve all these problems, but the techniques for doing it are tricky and vary widely. The simplest method is for the spot in the gel to be cut out with a razor blade, transferred into a vial where it is subjected to an enzyme such as trypsin which cuts the protein into peptides. The peptides are then transferred into a mass spectrometer where their mass is determined. The masses of these peptides form a peptide mass map which can be compared to the protein databases for identification of the protein.<\/p>\n

While peptide mass fingerprinting works quite well for identifying proteins >80% of the time, it has three major failings. First, proteins that are resistent to digestion, for whatever reason, are often missed. Second, due to overlap in the databases, some proteins are very difficult to distinguish. Third, the protein is usually identified on the basis of 3-5 peptide masses that match those predicted by the database, but the other peptides (often >15-20) are not detected and several peaks at anomalous masses are often detected. The first problem can be addressed somewhat by “Top-Down” sequencing method promoted by F. W. McLafferty<\/a>. The second can be addressed by improving the mass accuracy of the mass spectrometer to remove the ambiguity. The third is usually due to mutations in the protein, modification of the protein by addition of glycans or other post-translational modifications, or lack of ionization efficiency for the non detected peptides, e.g. because it is hydrophobic or insoluble.<\/p>\n

Instrument development is underway to address each of these problems. In addition, significant progress is needed in the automation of the instruments in order to allow the average biochemist access to the technology.<\/p>\n

 <\/p>\n","protected":false},"excerpt":{"rendered":"

Introduction to Proteomics Proteomics, broadly speaking, is the study of proteins, usually in the context of identity, quantity, and structure of the protein and its modifications in a cell or tissue as a function of time or some external change. The most common method for generating maps of proteins in a cell at a particular […]<\/p>\n","protected":false},"author":2,"featured_media":0,"parent":12,"menu_order":1,"comment_status":"closed","ping_status":"closed","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/pages\/13"}],"collection":[{"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/comments?post=13"}],"version-history":[{"count":1,"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/pages\/13\/revisions"}],"predecessor-version":[{"id":1012,"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/pages\/13\/revisions\/1012"}],"up":[{"embeddable":true,"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/pages\/12"}],"wp:attachment":[{"href":"https:\/\/www.bumc.bu.edu\/ftms\/wp-json\/wp\/v2\/media?parent=13"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}