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Analytical Chemistry

Proteomics Takes The Middle Road

Protease digests proteins into large peptides for middle-down proteomics

by Celia Henry Arnaud
June 25, 2012 | A version of this story appeared in Volume 90, Issue 26

Researchers led by Northwestern University’s Neil L. Kelleher report a protease that digests proteins into large peptides for mass spectrometric analysis in “middle down” proteomics (Nat. Methods, DOI: 10.1038/nmeth.2074). Bottom-up proteomics uses trypsin or other proteolytic enzymes to digest proteins into small peptides that make identifying proteins or posttranslational modifications difficult, especially in complex mixtures. Performance in top-down proteomics, which involves analyzing intact proteins, decreases as the proteins increase in size. Middle-down proteomics addresses some of the shortcomings of these two conventional proteomics methods. Kelleher and coworkers used an outer membrane protease T (OmpT) from Escherichia coli to cleave proteins into peptides about 5 to 20 kilodaltons in size. By interrogating peptides smaller than 15 kDa produced during OmpT digestion of proteins from human cancer cells, the researchers identified 3,697 peptides from 1,038 proteins. They found that OmpT has a preference for dibasic sites with two positively charged amino acids, lysine and arginine, at its cleavage recognition sites. The resulting peptides were large enough for the researchers to differentiate closely related protein isoforms and to detect posttranslational modifications.


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