Sampling the N-terminal proteome of human blood

Wildes, David; Wells, James A.

doi:10.1073/pnas.0914495107

Cited by 107 publications

(95 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As in other studies (4), we created a master set containing the most abundant cleavage site for each protein, based on the greatest number of supporting MS/MS spectra. As has been noted in other N-terminomics studies (14,35,36), we observe many proteins with "ladders" of N-termini at consecutive positions (supplemental Fig. S6), which might indicate either artifact (e.g.…”

Section: Identification Of Cleaved N-termini From Mousementioning

confidence: 57%

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast

Calvo

Julien

Clauser

et al. 2017

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of ϳ20 -60 amino acids, a net charge between ؉3 to ؉6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, ϳ80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.063818, 512-523, 2017.Mitochondria are ancient bacterium-derived organelles essential for eukaryotic life. A double membrane divides mitochondria into distinct compartments (matrix, inner membrane, intermembrane space, and outer membrane) that carry out specialized cellular processes, including oxidative phosphorylation, iron-sulfur cluster biogenesis, and a myriad biosynthetic pathways. Mammalian mitochondria contain a tiny genome encoding 13 proteins, whereas the remaining ϳ1200 proteins (1, 2) are encoded in the nucleus and imported into the organelle. Although there are several different mechanisms that can target proteins to the mitochondrion, the predominant mechanism is via an N-terminal presequence that directs import through the double membrane, which is subsequently cleaved to produce the mature, functional protein (3).To date, most of our understanding of mitochondrial protein import and processing has been elucidated using bakers' yeast as a model system. As reviewed recently (3), the canonical import pathway involves an amphipathic ␣-helical N-terminal sequence that directs import through the TOMM-TI...

show abstract

Section: Identification Of Cleaved N-termini From Mousementioning

confidence: 57%

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast

Calvo

Julien

Clauser

et al. 2017

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

show abstract

“…Our analysis of proteolysis substrates is based on a previously described N-terminal isolation platform that compares cells or lysates before and after initiating a proteolytic process (3,9,10). Briefly, free N termini in lysates are enzymatically labeled with a biotinylated peptide ester, captured on neutravidin beads, and trypsinized to produce N-terminal peptides.…”

Section: Resultsmentioning

confidence: 99%

Global kinetic analysis of proteolysis via quantitative targeted proteomics

Agard

Mahrus

Trinidad

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

124

152

View full text Add to dashboard Cite

Mass spectrometry-based proteomics is a powerful tool for identifying hundreds to thousands of posttranslational modifications in complex mixtures. However, it remains enormously challenging to simultaneously assess the intrinsic catalytic efficiencies (k cat ∕K M ) of these modifications in the context of their natural interactors. Such fundamental enzymological constants are key to determining substrate specificity and for establishing the timing and importance of cellular signaling. Here, we report the use of selected reaction monitoring (SRM) for tracking proteolysis induced by human apoptotic caspases-3, -7, -8, and -9 in lysates and living cells. By following the appearance of the cleaved peptides in lysate as a function of time, we were able to determine hundreds of catalytic efficiencies in parallel. Remarkably, we find the rates of substrate hydrolysis for individual caspases vary greater than 500-fold indicating a sequential process. Moreover, the rank-order of substrate cutting is similar in apoptotic cells, suggesting that cellular structures do not dramatically alter substrate accessibility. Comparisons of extrinsic (TRAIL) and intrinsic (staurosporine) inducers of apoptosis revealed similar substrate profiles, suggesting the final proteolytic demolitions proceed by similarly ordered plans. Certain biological processes were rapidly targeted by the caspases, including multiple components of the endocyotic pathway and miRNA processing machinery. We believe this massively parallel and quantitative label-free approach to obtaining basic enzymological constants will facilitate the study of proteolysis and other posttranslational modifications in complex mixtures.apoptosis | caspase | enzymology | mass spectrometry | selected reaction monitoring A poptosis is a form of programmed cell death that serves to eliminate unnecessary, infected, or tumorigenic cells from eukaryotic organisms. While many intrinsic and extrinsic stimuli can initiate apoptosis, these ultimately converge on the activation of a related family of aspartate-specific cysteine proteases, the caspases, that execute widespread proteolysis and induce noninflammatory death (1). We and others have surveyed N termini that occur in apoptotic cells and collectively reported more than 1,000 caspase-derived cleavages (2-5). This explosion of proteomic data has defined a vast array of caspase substrates proteolyzed during apoptosis. While these data identify caspase targets, and in some cases the sites of proteolysis, they fail to reveal the relative rates of cleavage, a parameter necessary to establish the order of proteolytic events and their importance in extracts and intact cells.The recent application of selected reaction monitoring (SRM) methods, traditionally used for metabolite identification, to proteomic studies has enabled the simultaneous label-free quantification of hundreds of peptides (6, 7). Our development of a N-terminal enrichment platform (3) is ideally suited to the application of SRM to apoptotic proteolysis. Using this platfor...

show abstract

“…Surprisingly, there are only 45 sites in the 2900 noncaspase, nontryptic (not cleaved after lysine or arginine) endoproteolytic events (residue 66ϩ) that are present in the MEROPS database (release 9.6), and only 10 of these sites are annotated as "physiological" cleavages in MEROPS (supplemental Table S4). Interestingly, there is little exoproteolysis of these intracellular proteins, whereas laddering produced by sequential exoproteolysis was observed in 24% of all proteins identified in a subtiligase-based study of human serum (9).…”

Section: Discussionmentioning

confidence: 99%

“…We have developed a positive enrichment method in which the ␣-amines of intracellular (8) or extracellular proteins (9) can be specifically and directly tagged and captured, without pretreatment or protection, using subtiligase, an engineered peptide ligase (Fig. 1A) (10,11).…”

mentioning

confidence: 99%

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

Crawford

Seaman²,

Agard³

et al. 2013

Molecular & Cellular Proteomics

Self Cite

130

173

View full text Add to dashboard Cite

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

show abstract

Sampling the N-terminal proteome of human blood

Cited by 107 publications

References 43 publications

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast

Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast

Global kinetic analysis of proteolysis via quantitative targeted proteomics

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

Contact Info

Product

Resources

About