In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation

Hanna, Rawad; Rozenberg, Andrey; Saied, Layla; Ben-Yosef, Daniel; Lavy, Tali; Kleifeld, Oded

doi:10.1016/j.mcpro.2023.100584

Cited by 9 publications

(1 citation statement)

References 89 publications

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“…COFRADIC (combined fractional diagonal chromatography) (10) and TAILS (terminal amine isotopic labeling of substrates) (11) are widely known as N-terminal peptide isolation methods. Even recently, new methods for N-terminal peptide isolation have been reported (12). However, all of these methods require complex and time-consuming procedures involving chemical modifications that block amines, and the efficiency and selectivity of the chemical modifications are also issues.…”

Section: Introductionmentioning

confidence: 99%

One-step N-terminomics based on isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography

Morikawa,

Nishida,

Imami

et al. 2024

Preprint

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We have developed a one-step isolation method for protein N-terminal peptides from LysargiNase digests by pipette tip-based strong cation exchange (SCX) chromatography. This CHAMP-N (CHromatographic AMplification of Protein N-terminal peptides) method using disposable and parallel-processable SCX tips instead of conventional HPLC SCX columns facilitates simple, sensitivie and high-throughput N-terminomic profiling without sacrificing identification numbers and selectivity achieved by the HPLC-based method. By applying the CHAMP-N method to HEK293T cells, we identified novel cleavage sites for signal and transit peptides, and non-canonical translation initiation sites. Finally, for proteome-wide terminomics, we have established a simple and comprehensive N- and C-terminomics platform based on three different tip-based approaches including CHAMP-N, in which protease digestion and one-step isolation by tip LC are commonly used to achieve complementary terminome coverage.In BriefLarge-scale isolation of protein N-terminal peptides from LysargiNase digests was achieved by one-step strong cation exchange chromatography with disposable and parallel-processable pipette-tip columns. Novel cleavage sites for signal or transit peptides and non-canonical translation initiation sites were identified by this tip-based method. Together with two other methods similarly consisting of protease digestion and one-step tip LC, we have established a simple and comprehensive N- and C-terminomics platform based on CHAMP (chromatographic amplification of protein terminal peptides) approaches.HighlightsOne-step isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography.Highly efficient isolation of protein N-terminal peptides by tip LC comparable to HPLC.Identifying novel post-translational cleavage sites and non-canonical translation initiation sites.Complementary terminome coverage by the three CHAMP methods consisting of protease digestion and one-step tip LC.

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Section: Introductionmentioning

confidence: 99%

One-step N-terminomics based on isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography

Morikawa,

Nishida,

Imami

et al. 2024

Preprint

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TermineR: Extracting information on endogenous proteolytic processing from shotgun proteomics data

Cosenza‐Contreras,

Seredynska,

Vogele

et al. 2024

Proteomics

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State‐of‐the‐art mass spectrometers combined with modern bioinformatics algorithms for peptide‐to‐spectrum matching (PSM) with robust statistical scoring allow for more variable features (i.e., post‐translational modifications) being reliably identified from (tandem‐) mass spectrometry data, often without the need for biochemical enrichment. Semi‐specific proteome searches, that enforce a theoretical enzymatic digestion to solely the N‐ or C‐terminal end, allow to identify of native protein termini or those arising from endogenous proteolytic activity (also referred to as “neo‐N‐termini” analysis or “N‐terminomics”). Nevertheless, deriving biological meaning from these search outputs can be challenging in terms of data mining and analysis. Thus, we introduce TermineR, a data analysis approach for the (1) annotation of peptides according to their enzymatic cleavage specificity and known protein processing features, (2) differential abundance and enrichment analysis of N‐terminal sequence patterns, and (3) visualization of neo‐N‐termini location. We illustrate the use of TermineR by applying it to tandem mass tag (TMT)‐based proteomics data of a mouse model of polycystic kidney disease, and assess the semi‐specific searches for biological interpretation of cleavage events and the variable contribution of proteolytic products to general protein abundance. The TermineR approach and example data are available as an R package at https://github.com/MiguelCos/TermineR.

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Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry

Stockert,

Baeta,

Huesgen

2024

Methods in Enzymology

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In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation

Cited by 9 publications

References 89 publications

One-step N-terminomics based on isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography

One-step N-terminomics based on isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography

TermineR: Extracting information on endogenous proteolytic processing from shotgun proteomics data

Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry

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