Selecting protein N-terminal peptides by combined fractional diagonal chromatography

Staes, An; Impens, Francis; Damme, Petra Van; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

doi:10.1038/nprot.2011.355

Cited by 161 publications

(184 citation statements)

References 51 publications

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“…[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In addition to providing unbiased information about which proteins are cleaved, in some cases, these experiments locate the precise sites and quantify the rates of cleavage.…”

Section: -5mentioning

confidence: 99%

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues

et al. 2016

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Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity. Human caspases are a family of 12 homologous intracellular proteases known for driving cellular state changes such as apoptosis and differentiation, as well as inflammatory responses. Caspases are cysteine-class proteases named for their signature ability to cleave after aspartate residues, or P1 is aspartate using the Schetchter and Berger notation. 1,2Synthetic peptide profiling for purified caspases show distinctive subsite preferences extending from P1 to P4 or P5. 3-5Sequence conservation and signal pathway analyses have further grouped the proteases into apoptotic initiators (−2, − 8, − 9 and − 10), apoptotic executioners (−3, − 6 and − 7), regulators of inflammation (−1, − 4, − 5 and − 12) and keratinocyte differentiation − 14).The past decade has seen a significant advancement in the use of LC-MS to define the spectrum of natural proteins cleaved by caspases in cells. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In addition to providing unbiased information about which proteins are cleaved, in some cases, these experiments locate the precise sites and quantify the rates of cleavage.13-17 Using the subtiligasebased N-terminomics approach, we identified more than 1700 aspartate cleavage eve...

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Section: -5mentioning

confidence: 99%

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues

et al. 2016

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“…After blocking the activity of endogenous caspases by iodoacetamide (42) (39,43). As part of the N-terminal COFRADIC protocol, differential acetylation was then used to distinguish further between the two light-labeled samples (N-hydroxysuccinimide trideutero-acetate was used for the caspase-2, -3, and -7 samples and N-hydroxysuccinimide acetate for the control sample) and to distinguish between in vitro and in vivo N-acetylation of the caspase-treated samples, the former indicative for proteolytic events (43). Recombinant human active caspase-2, -3, and -7 were purchased from R&D Systems (Minneapolis, MN) (43).…”

Section: Silac Labeling and N-terminal Cofradic Setup-silacmentioning

confidence: 99%

“…F-12K medium was supplemented with 0.3 mM L-arginine (15% of the standard concentration, at which the conversion of L-arginine to proline was not observed) using [ 12 After SILAC labeling, A549 cells were harvested and lysed as described (42). After blocking the activity of endogenous caspases by iodoacetamide (42) (39,43). As part of the N-terminal COFRADIC protocol, differential acetylation was then used to distinguish further between the two light-labeled samples (N-hydroxysuccinimide trideutero-acetate was used for the caspase-2, -3, and -7 samples and N-hydroxysuccinimide acetate for the control sample) and to distinguish between in vitro and in vivo N-acetylation of the caspase-treated samples, the former indicative for proteolytic events (43).…”

Section: Silac Labeling and N-terminal Cofradic Setup-silacmentioning

confidence: 99%

Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

Wejda

Impens

Takahashi

et al. 2012

Journal of Biological Chemistry

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Background:To study the potential role of caspase-2, we determined its cleavage site specificity and compared it with that of caspase-3 and -7. Results: N-terminal COFRADIC analysis of native proteomes revealed that caspase-2, -3, and -7 have overlapping specificities. Conclusion: Caspase-2, -3, and -7 share nearly indistinguishable cleavage site specificity. Significance: This finding suggests that caspase-2 has a proapoptotic function.

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“…Free ␣-amines occur on some proteins that are never N-terminally acetylated, and can also be regenerated by signal or transit peptide removal during protein trafficking, and endo-or exoproteolysis during protein maturation and signaling. Thus, there has been considerable effort to develop unbiased proteomic methods to characterize the ␣-aminome in healthy and diseased states (3)(4)(5)(6)(7)(8).…”

mentioning

confidence: 99%

The DegraBase: A Database of Proteolysis in Healthy and Apoptotic Human Cells

Crawford

Seaman²,

Agard³

et al. 2013

Molecular & Cellular Proteomics

122

170

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Proteolysis is a critical post-translational modification for regulation of cellular processes. Our lab has previously developed a technique for specifically labeling unmodified protein N termini, the ␣-aminome, using the engineered enzyme, subtiligase. Here we present a database, called the DegraBase (http://wellslab.ucsf.edu/degrabase/), which compiles 8090 unique N termini from 3206 proteins directly identified in subtiligase-based positive enrichment mass spectrometry experiments in healthy and apoptotic human cell lines. We include both previously published and unpublished data in our analysis, resulting in a total of 2144 unique ␣-amines identified in healthy cells, and 6990 in cells undergoing apoptosis. The N termini derive from three general categories of proteolysis with respect to cleavage location and functional role: translational N-terminal methionine processing (ϳ10% of total proteolysis), sites close to the translational N terminus that likely represent removal of transit or signal peptides (ϳ25% of total), and finally, other endoproteolytic cuts (ϳ65% of total). Induction of apoptosis causes relatively little change in the first two proteolytic categories, but dramatic changes are seen in endoproteolysis. For example, we observed 1706 putative apoptotic caspase cuts, more than double the total annotated sites in the CASBAH and MEROPS databases. In the endoproteolysis category, there are a total of nearly 3000 noncaspase nontryptic cleavages that are not currently reported in the MEROPS database. These studies significantly increase the annotation for all categories of proteolysis in human cells and allow public access for investigators to explore interesting proteolytic events in healthy and apoptotic human cells. Molecular & Cellular

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Selecting protein N-terminal peptides by combined fractional diagonal chromatography

Cited by 161 publications

References 51 publications

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues

Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

The DegraBase: A Database of Proteolysis in Healthy and Apoptotic Human Cells

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