Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

Xu, Guoqiang; Shin, Sung Bin Y.; Jaffrey, Samie R.

doi:10.1073/pnas.0908958106

Cited by 92 publications

(81 citation statements)

References 40 publications

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“…According to UniProtKB (http://www.uniprot.org/uniprot/Q16740), CLPP is a 277 amino acids long protein with a 56 amino acid long transit peptide at its N-terminal [18]. In the presence of ATP and magnesium, CLPP has peptidase activity and can hydrolyze larger proteins to smaller peptides [19][20][21].…”

Section: Discussionmentioning

confidence: 99%

Exome analysis identified a novel missense mutation in the CLPP gene in a consanguineous Saudi family expanding the clinical spectrum of Perrault Syndrome type-3

Ahmed

Jelani

Alrayes

et al. 2015

Journal of the Neurological Sciences

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Section: Discussionmentioning

confidence: 99%

Exome analysis identified a novel missense mutation in the CLPP gene in a consanguineous Saudi family expanding the clinical spectrum of Perrault Syndrome type-3

Ahmed

Jelani

Alrayes

et al. 2015

Journal of the Neurological Sciences

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“…However, up to 50 to 100 mg of protein is required for each sample analysis, which can be very limiting. In a clever use of Edman chemistry, Xu et al used chemical labeling of the ␣-amine of proteins (N-CLAP) using phenyl isothiocyanate to block all primary amines (34). Similar to Edman degradation, treatment with trifluoroacetic acid triggers cyclization of phenyl-isothiocyanate-modified ␣-amines specifically, resulting in peptide bond cleavage after the first amino acid.…”

Section: Methods For Enriching Protein N Termini-n-terminalmentioning

confidence: 99%

Proteolytic Post-translational Modification of Proteins: Proteomic Tools and Methodology

Rogers

Overall²

2013

Molecular & Cellular Proteomics

140

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Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini. Here we provide an overview of contemporary proteomics-based methods, including specific quantitation, data analysis, and curation considerations, and highlight exciting new and emerging applications within these fields enabling in vivo analysis of proteolytic events. Proteolysis involves the breakdown of proteins into smaller polypeptides or amino acids through the hydrolysis of peptide bonds by a protease. This represents a remarkably significant, but often underappreciated, post-translational modification (PTM) 1 in that is it irreversible yet also ubiquitous. Consequently, the functional sequence of a protein can very rarely be predicted from its transcript, as proteolysis products form new (neo-) N and C termini. These cleavage events, or proteolytic processing events, can result in activation, inactivation, completely altered protein function, and even excision of "neo-proteins" with growth factor activity from an extracellular matrix parent molecule, and they regulate a vast array of biological processes (1). These include DNA replication, cell cycle progression, cell proliferation, and cell death, as well as pathological processes such as inflammation, cancer, arthritis, and cardiovascular disease. For example, in protein synthesis and maturation, precise selective removal of the N-terminal methionine and the signal peptide is essential for correct protein maturation and secretion. In some proteins, scission of the chain forms a molecule with four termini when linked by disulfide bridges. Through the removal of signal, nuclear, and mitochondrial localization sequences and ectodomain shedding, proteases regulate protein localization, and in viral infection, via cleavage of pre-and pro-domains and polyprotein processing, inactive proteins are converted into their active form(s), are inactivated, or change receptor-binding affinity. Thus, proteolysis is involved in much more than the mere degradation and turnover of proteins, important though these processes are in homeostasis.Proteases exist in all orders of life and constitute one of the largest enzyme families in humans (2), and more than 30 drugs targeting these enzymes are currently approved for clinical use (3). However, in order to fully comprehend the cellular function(s) of a given protease, one must have knowledge of the proteins processed by that protease, as well as the functions of these substrates and specific processing events. This is currently far from the case, as half of all human proteases have no known substrates (4). Degradomic...

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“…Positive selection is based on the use of highly selective enzymes or chemical reactions targeting (typically by biotinylation) the free α-amino group of proteins and their proteolytic fragments, thus directly selecting for N-terminal peptides [4][5][6] . In contrast, negative procedures, such as N-terminal combined fractional diagonal chromatography (COFRADIC), target and remove internal peptides 2,3 .…”

mentioning

confidence: 99%

Selecting protein N-terminal peptides by combined fractional diagonal chromatography

et al. 2011

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In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

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Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

Cited by 92 publications

References 40 publications

Exome analysis identified a novel missense mutation in the CLPP gene in a consanguineous Saudi family expanding the clinical spectrum of Perrault Syndrome type-3

Exome analysis identified a novel missense mutation in the CLPP gene in a consanguineous Saudi family expanding the clinical spectrum of Perrault Syndrome type-3

Proteolytic Post-translational Modification of Proteins: Proteomic Tools and Methodology

Selecting protein N-terminal peptides by combined fractional diagonal chromatography

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