Identification of cRAP Proteins in Mass Spectrometry

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Thursday 10/20/2016

Since the early days of mass spectrometry-based proteomics protein contamination has been observed with proteins, like keratins, constantly identified in qualitative results. In the case of keratins, these proteins originate from the hair, skin, fingernails, and clothing of anyone handling the sample or associated with the sample. In analysis of human samples, signal produced in the mass spectrometer from keratin can be correctly identified through protein database searching since keratin proteins are components of the human protein database.

Potential issues arise with a protein database that does not contain keratin in the database, say a yeast database. Spectra from the mass spectrometer corresponding to keratins are now identified to yeast proteins in the database. It is hoped that validation software will remove these misidentified proteins; however, it is impossible to know if this happens. To circumvent this problem, keratin proteins would be added to the yeast database to ensure mass spectrometry signals originating from keratin are correctly identified as such and not as yeast proteins, thus preventing a source of false discovery.

While keratin was used in the example above, the Global Proteome Machine (www.thegpm.org) has developed a common Repository of Adventitious Proteins (cRAP; pronounced "cee-RAP") that are frequently identified in proteomics experiments. These proteins are 1) common laboratory proteins. 2) proteins added through dust or physical contact, and 3) proteins used as standards for mass spectrometry. It is standard practice for the Fred Hutch Proteomics Facility to appended protein databases with cRAP to ensure potentially contaminating proteins are identified correctly and, in turn, control the protein identification false discovery rate. Therefore, if you have ever wondered why bovine albumin was identified in your yeast lysate, it is because the protein was from cRAP.

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Rajesh K. Uthamanthil, DVM, PhD, DACLAM

Interim Leader, Shared Resources
Director, Comparative Medicine