Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.
We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.
From the ‡Institute of Molecular Medicine and Cell Research.
Martin L
ML
Biniossek
§Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria;
Melanie
M
Niemer
¶Department of Biochemistry.
Ken
K
Maksimchuk
From the ‡Institute of Molecular Medicine and Cell Research.
Bettina
B
Mayer
Julian
J
Fuchs
**Central Institute for Engineering, Electronics and Analytics, ZEA-3 Analytics, Forschungszentrum Jülich, 52425 Jülich, Germany;
Pitter F
PF
Huesgen
¶Department of Biochemistry.
Dewey G
DG
McCafferty
‡‡Department of Chemistry, Duke University, Medical Center, Durham, NC 27708, USA; Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria;
Boris
B
Turk
§§Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia; Department of Human Genetics and Center of Human and Molecular Biology, Medical Faculty, University of Saarland Homburg, Germany;
Guenther
G
Fritz
¶¶Institute of Neuropathology.
Jens
J
Mayer
***Institute for Medical Microbiology and Hygiene.
Georg
G
Haecker
§Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria;
Lukas
L
Mach
From the ‡Institute of Molecular Medicine and Cell Research, ‡‡‡BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany; §§§German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany oliver.schilling@mol-med.uni-freiburg.de.
Oliver
O
Schilling
D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D010447:Peptide Hydrolases / Q000378:metabolism*; D019151:Peptide Library; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D019151:Peptide Library; D020543:Proteome; D010447:Peptide Hydrolases