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A protease for 'middle-down' proteomics


We developed a method for restricted enzymatic proteolysis using the outer membrane protease T (OmpT) to produce large peptides (>6.3 kDa on average) for mass spectrometry–based proteomics. Using this approach to analyze prefractionated high-mass HeLa proteins, we identified 3,697 unique peptides from 1,038 proteins. We demonstrated the ability of large OmpT peptides to differentiate closely related protein isoforms and to enable the detection of many post-translational modifications.

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Figure 1: OmpT-based platform for middle-down proteomics and characterization of OmpT peptides from digestion of a standard protein.
Figure 2: Proteotypic OmpT peptides, peptide size distribution and iceLogo of OmpT recognition site.


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We sincerely thank current and former members of the Kelleher and Sweedler research groups, especially B. Evans, P. Chu, P. Compton, A. Catherman, S. Sweet, I. Ntai, J. Lee, A. Vellaichamy and K. Catherman, for technical support and insightful suggestions, P. Yau and B. Imai from the Protein Sciences Facility at the University of Illinois for the synthesis of the fluorogenic substrate and M. Burke for the access to a fluorimeter. The project was supported by the US National Institutes of Health through awards R01 GM067193, P30 DA018310 and F30 DA026672, by the National Science Foundation through award DMS 0800631 and by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust.

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Authors and Affiliations



C.W. designed research, conducted experiments, analyzed data and wrote the paper; J.C.T., L.Z., K.R.D. and B.P.E. analyzed data; M.L., K.R.D. and D.R.A. conducted cell culture; P.M.T. analyzed data and wrote the paper; J.V.S. interpreted data and wrote the paper; N.L.K. designed research, interpreted data and wrote the paper.

Corresponding author

Correspondence to Neil L Kelleher.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Note (PDF 2307 kb)

Supplementary Table 1

Identified unique OmpT peptide lists and unique protein counts from Protein Center report. Unique OmpT peptides identified from absolute mass and biomarker searches are listed separately as well as in a combined list after removing the redundant overlapped hits from both search modes. (XLS 9183 kb)

Supplementary Table 2

Pooled unique OmpT peptide identifications from the nanoLC-MS/MS injections for the CID and ETD comparisons. (XLS 2739 kb)

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Wu, C., Tran, J., Zamdborg, L. et al. A protease for 'middle-down' proteomics. Nat Methods 9, 822–824 (2012).

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