A streamlined platform for high-content functional proteomics of primary human specimens

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Abstract

Achieving information content of satisfactory breadth and depth remains a formidable challenge for proteomics. This problem is particularly relevant to the study of primary human specimens, such as tumor biopsies, which are heterogeneous and of finite quantity. Here we present a functional proteomics strategy that unites the activity-based protein profiling and multidimensional protein identification technologies (ABPP-MudPIT) for the streamlined analysis of human samples. This convergent platform involves a rapid initial phase, in which enzyme activity signatures are generated for functional classification of samples, followed by in-depth analysis of representative members from each class. Using this two-tiered approach, we identified more than 50 enzyme activities in human breast tumors, nearly a third of which represent previously uncharacterized proteins. Comparison with cDNA microarrays revealed enzymes whose activity, but not mRNA expression, depicted tumor class, underscoring the power of ABPP-MudPIT for the discovery of new markers of human disease that may evade detection by other molecular profiling methods.

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Figure 1: Integration of the ABPP and MudPIT methods for high-content functional proteomics of primary human specimens.
Figure 2: Enzyme activity signatures of primary human breast tumors and normal breast tissue.
Figure 3: Comparison of enzyme activity levels measured by 1DE-ABPP and MudPIT-ABPP.
Figure 4: Comparison of activity and mRNA expression levels for FAP and KIAA1363.

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Acknowledgements

We thank D. Cociorva for help with DTASelect and Sequest. This work was supported by the US National Institutes of Health grants CA087660 (to B.F.C.) and HG00030 (to M.N.), the California Breast Cancer Research Foundation (B.F.C. and S.S.J.), the Susan G. Komen Breast Cancer Foundation (B.Q.W.) and the Skaggs Institute for Chemical Biology.

Author information

Correspondence to Benjamin F Cravatt.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Structures of fluorophosphonate (FP) ABPP probes. (PDF 358 kb)

Supplementary Fig. 2

Enzyme activity profiles for primary human breast tumor and normal breast specimens determined by 1D-gel ABPP. (PDF 2922 kb)

Supplementary Fig. 3

Representative gel showing molecular mass annotations of enzyme activities used in the clustering analysis shown in Figure 2b of the manuscript. (PDF 2310 kb)

Supplementary Fig. 4

Gene expression profiles for differentially expressed enzyme activities in primary human breast tumor and normal breast specimens. (PDF 313 kb)

Supplementary Table 1

Clinical data on breast tumors. (PDF 67 kb)

Supplementary Table 2

A complete list of the FP-labeled serine hydrolase activities identified in primary human breast tumor and normal breast specimens. (PDF 66 kb)

Supplementary Table 3

Reproducibility of enzyme activity data for ER(-)/PR(-) breast tumor BC48. (PDF 42 kb)

Supplementary Methods (PDF 275 kb)

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Jessani, N., Niessen, S., Wei, B. et al. A streamlined platform for high-content functional proteomics of primary human specimens. Nat Methods 2, 691–697 (2005) doi:10.1038/nmeth778

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