Protease cleavage site fingerprinting by label‐free in‐gel degradomics reveals pH ‐dependent specificity switch of legumain

Abstract

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel-based label-free proteomic approach (DIPPS-direct in-gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in-gel digestion of the gel-separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length-limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase-7, and legumain) to broadly specific (matrix-metalloproteinase-3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH-dependent change in the specificity of this protease, further supporting its broad applicability.

Novel Apoptotic Mediators Identified by Conservation of Vertebrate Caspase Targets

“…Interestingly, around 5% of vertebrate cleavage site orthologs have glutamate in the P1 position instead of aspartate (Table 1). Previous in vitro reports have shown that caspases-3, -6, and -7 are able to accommodate P1 glutamate in the active site and cleave substrates with P1 E cut sites with the same affinity but with a twofold slower rate than substrates with aspartate in the same position [13,37]. The same study by the Wells group demonstrated that substitution of P1 glutamate by aspartate actually results in a higher rate of protein cleavage by caspases.…”

Mapping specificity, entropy, allosteric changes and substrates in blood proteases by a high-throughput protease screen

“…In the second step, the protease-generated peptides can be identified with any tandem mass spectra search tool. In our implementation, we used the Andromeda 30 search engine in MaxQuant 31 using unspecific database search parameters as described elsewhere 21 . Importantly, we searched the data with a reduced database (HTPS_DB.fasta) generated from proteins identified in Trypsin, Lys-C, Asp-N, Glu-C and Chymotrypsin samples.…”

“…Relevant techniques to identify protease substrates include COFRADIC (combined fractional diagonal chromatography) 10,11 , ChaFraDIC (charge-based fractional diagonal chromatography) 12 , PICS (proteomic identification of protease cleavage sites) 13,14 and TAILS (terminal amine isotopic labeling of substrates) 15,16 which are reviewed elsewhere [17][18][19] . More recently, workflows like FPPS (fast profiling of protease specificity) 20 and especially label-free degradomic workflows like DIPPS (direct in-gel profiling of protease specificity) 21 and ChaFraTip (ChaFraDIC performed in a pipet tip format) 22 made protease characterization easier and more accessible by describing simplified workflows and omitting extensive fractionation or labeling steps. Furthermore, DIPPS 21 and ChaFraTip 22 can simultaneously map prime and non-prime substrate sites by sequencing the protease-generated peptides.…”

“…More recently, workflows like FPPS (fast profiling of protease specificity) 20 and especially label-free degradomic workflows like DIPPS (direct in-gel profiling of protease specificity) 21 and ChaFraTip (ChaFraDIC performed in a pipet tip format) 22 made protease characterization easier and more accessible by describing simplified workflows and omitting extensive fractionation or labeling steps. Furthermore, DIPPS 21 and ChaFraTip 22 can simultaneously map prime and non-prime substrate sites by sequencing the protease-generated peptides. In spite of these developments, several limitations remain which limit the throughput, costeffectiveness or physiological relevance of these assays.…”

The Acute Phase Response Is a Prominent Renal Proteome Change in Sepsis in Mice

“…Four kidney samples selected as most representative from each treatment group and four control kidney samples were processed for mass spectrometry analysis as described before [79] with a slightly modified protocol. Frozen tissues were cut into pieces of about 30 mg on dry ice to prevent thawing.…”