Protein amino-termini and how to identify them

Bogaert, Annelies; Gevaert, Kris

doi:10.1080/14789450.2020.1821657

Cited by 12 publications

(10 citation statements)

References 98 publications

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“…These techniques mostly rely on the bottom-up proteomics, in which a site-specific externally added protease digests proteomes to generate peptides before chromatographic separation and tandem MS (MS/MS) analysis, in contrast to the top-down proteomics approach, which directly analyzes intact proteins with all retained modifications by MS (Aebersold and Mann, 2016). Various proteomics technologies for Nand C-terminal analysis have been recently comprehensively described (reviewed in Tanco et al, 2015;Demir et al, 2018;Kaleja et al, 2019;Luo et al, 2019;Perrar et al, 2019;Bogaert and Gevaert, 2020;Kaushal and Lee, 2021;Mintoo et al, 2021). Due to some technical difficulties (e.g., poor ionization efficiencies of protein C-termini, low yielding carboxyl group labeling strategies), C-terminomics technologies are, however, less successfully implemented and widely used, in comparison to the N-terminomics methods, which in recent years have become an indispensable tool in positional proteomics (Eckhard et al, 2016).…”

Section: Methods For Characterization Of Mitochondrial Protein N-term...mentioning

confidence: 99%

“…A positive selection strategy relies on a direct enrichment of N-terminal peptides from the peptide mixture [e.g., the Subtiligase and Chemical Enrichment of Protease Substrates (CHOPS) methods]. In the negative selection methods, the N-terminal peptides are enriched indirectly by the removal of undesired (internal, C-terminal) peptides (e.g., COFRADIC, ChaFRADIC, and TAILS methods) (Bogaert and Gevaert, 2020).…”

Section: Methods For Characterization Of Mitochondrial Protein N-term...mentioning

confidence: 99%

“…After isolation and denaturation of studied proteomes, cysteine residues are first reduced and alkylated. This sample preparation step is commonly used in many, if not all, N-terminomics studies (Bogaert and Gevaert, 2020). Subsequently, all primary amines [α-and ε-amines of protein N-terminus and lysine (Lys) residues] are labeled using N-hydroxysuccinimide (NHS) acetate ester in N-acylation reaction.…”

Section: Combined Fractional Diagonal Chromatographymentioning

confidence: 99%

“…After pooling the samples and digesting them by trypsin, which hydrolyzes the peptide bonds C-terminal of Arg only, the peptide mixtures are separated by first SCX chromatography run at a pH of 2.7 (Figure 3B). After the first SCX run, which allows for selective separation of charged peptide fractions +1, +2, +3, +4, and >+4, all fractions are collected and treated with trideutero N-hydroxysuccinimide (d3-NHS) acetate (Venne et al, 2013;Bogaert and Gevaert, 2020). Only internal and C-terminal peptides with free α-amines are acetylated in this reaction, while the N-terminal peptides with blocked (i.e., labeled) α-amines remain unchanged.…”

Section: Charge-based Fractional Diagonal Chromatographymentioning

confidence: 99%

“…Since acetylation alters the charge of internal and C-terminal peptides, these modified peptides elute earlier in the second SCX chromatography run than during the first separation. The retention time of N-terminal peptides remains the same, allowing selective collection of native and neo-N-terminal peptides (Bogaert and Gevaert, 2020;Mintoo et al, 2021). The follow-up LC-MS/MS analysis provides the protease cleavage site and identifies processing protein substrates (Figure 3B).…”

Section: Charge-based Fractional Diagonal Chromatographymentioning

confidence: 99%

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Protein Processing in Plant Mitochondria Compared to Yeast and Mammals

2022

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Limited proteolysis, called protein processing, is an essential post-translational mechanism that controls protein localization, activity, and in consequence, function. This process is prevalent for mitochondrial proteins, mainly synthesized as precursor proteins with N-terminal sequences (presequences) that act as targeting signals and are removed upon import into the organelle. Mitochondria have a distinct and highly conserved proteolytic system that includes proteases with sole function in presequence processing and proteases, which show diverse mitochondrial functions with limited proteolysis as an additional one. In virtually all mitochondria, the primary processing of N-terminal signals is catalyzed by the well-characterized mitochondrial processing peptidase (MPP). Subsequently, a second proteolytic cleavage occurs, leading to more stabilized residues at the newly formed N-terminus. Lately, mitochondrial proteases, intermediate cleavage peptidase 55 (ICP55) and octapeptidyl protease 1 (OCT1), involved in proteolytic cleavage after MPP and their substrates have been described in the plant, yeast, and mammalian mitochondria. Mitochondrial proteins can also be processed by removing a peptide from their N- or C-terminus as a maturation step during insertion into the membrane or as a regulatory mechanism in maintaining their function. This type of limited proteolysis is characteristic for processing proteases, such as IMP and rhomboid proteases, or the general mitochondrial quality control proteases ATP23, m-AAA, i-AAA, and OMA1. Identification of processing protease substrates and defining their consensus cleavage motifs is now possible with the help of large-scale quantitative mass spectrometry-based N-terminomics, such as combined fractional diagonal chromatography (COFRADIC), charge-based fractional diagonal chromatography (ChaFRADIC), or terminal amine isotopic labeling of substrates (TAILS). This review summarizes the current knowledge on the characterization of mitochondrial processing peptidases and selected N-terminomics techniques used to uncover protease substrates in the plant, yeast, and mammalian mitochondria.

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