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Lauroylation-assisted workflow for N-terminomics with three biological replicates

ABSTRACT: Lauroylation-assisted N-terminomics for profiling protein N-termini, including ORF and non-ORF protein N-termini, using three biological replicates of etoposide-treated cholangiocarcinoma cells and exploiting negative selection.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ryoji Masui, Yoshikatsu Kanai

PROVIDER: PXD073552 | JPOST Repository | Fri May 01 00:00:00 BST 2026

REPOSITORIES: jPOST

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			Action	DRS
	20260108_KKU055_Eto_DM_ReC1-T1-2_02_C02_HpHC18_10.raw	Raw
	20260108_KKU055_Eto_DM_ReC1-T1-2_02_C02_HpHC18_15.raw	Raw
	20260108_KKU055_Eto_DM_ReC1-T1-2_02_C02_HpHC18_20.raw	Raw
	20260108_KKU055_Eto_DM_ReC1-T1-2_02_C02_HpHC18_25.raw	Raw
	20260108_KKU055_Eto_DM_ReC1-T1-2_02_C02_HpHC18_Elu.raw	Raw

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Publications

A Sensitive, Specific, and Cost-Effective Lauroylation-Assisted Workflow for Profiling Peptide-Level Protease Specificity Using Proteomic Identification of Cleavage Sites (PICS), with Applicability to Protein-Level N-Terminomics.

Okanishi Hiroki H Uno Isayuki I Nishimura Sho-Ichi SI Kanai Yoshikatsu Y Masui Ryoji R

Journal of proteome research 20260310 4

Proteases play crucial roles in numerous biological processes through specific protein cleavage, and their dysregulation has been implicated in various diseases. To better understand protease specificity, we developed a lauroylation-assisted proteomic identification of protease cleavage sites (PICS) workflow that labels and enriches targeted protease-generated neo-N-termini using economical reagents and standard laboratory equipment. The lauroylation enables both discrimination of the neo-N-term ...[more]

PMID: 41805451

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Lauroylation-assisted workflow for both PICS and N-terminomics

Project description:(1) Lauroylation-assisted workflow for PICS using E. coli proteome-derived tryptic peptide libraries which were blocked (succinylation or dimethylation). (2) Lauroylation-assisted N-terminomics to profile protein N-termini, ORF and non-ORF protein N-termini, using etoposide-treated cholangiocarcinoma cells by exploiting negative selection.

2026-05-01 | PXD067596 | JPOST Repository

Carboxypeptidase B-assisted charge-based fractional diagonal chromatography for deep screening of C-terminome

Project description:The determination of protein C-termini is of great significance for protein function annotation and proteolysis re-search. However, the progress of C-terminomics is still far behind its counterpart, N-terminomics, because of the low reactivity of the carboxyl group. Herein, we developed a negative selection strategy, termed carboxypeptidase B-assisted charge-based fractional diagonal chromatography (CPB-ChaFRADIC), to achieve a global C-terminome analysis. The highly reactive carboxypeptidase B cleavage was utilized to reduce the charge state of non-C-terminal peptides. Together with high-performance charge-based frac-tional diagonal chromatography, the C-terminal peptides could be isolated. Such a strategy was also applied for profiling C-termini from Escherichia coli cell lysates, and 441 canonical C-termini and 510 neo-C-termini originating from proteolytic processing were identified. These findings represent 2-fold and 5.8-fold that of identified C-termini via direct analysis, respectively. Using parallel digestion with trypsin and LysC, such a strategy enabled the identification of 604 canonical C-termini and 818 neo-C-termini, rep-resenting the largest C-terminome dataset of E. coli, and no deficiency in His/Lys/Arg-containing C-terminal peptides was observed. The presented CPB-ChaFRADIC strategy is therefore a highly efficient and unbiased strategy for large-scale C-terminome analysis. Furthermore, using the CPB-ChaFRADIC strategy, we identified 87 cleavage sites and 82 substrates of caspase-3 in Jurkat cells, demonstrating that the CPB-ChaFRADIC strategy shows great promise in promoting proteolysis research.

2020-05-25 | PXD018520 | Pride

Murine colon HUNTER N-terminomics

Project description:The mammalian lysosomal protease legumain is often dysregulated in pathophysiological conditions including inflammation, neurodegeneration, and cancer, yet its proteolytic targets are poorly defined. To profile protease substrates degradomics techniques typically employ enrichment strategies to select for sub-stoichiometric and low abundant peptides generated from proteolytic cleavage. However, recent advancements in degradomics techniques have revealed N-termini enrichment can be circumvented if peptide-based fractionation is employed, enabling simultaneous proteome and N-terminome analysis. Herein, we compare the previously published enrichment free N-terminomics approach using high-field asymmetric waveform ion mobility spectrometry (FAIMS) to offline basic reverse-phase (bRP) fractionation to assess the complementarity of these fractionation methods for simultaneous proteomic and degradomic analysis. While at the protein levels FAIMS and bRP provide access to overlapping proteomic coverage, at the N-terminal level each fractionation technique reveals unique cleavage information. Combining fractionation approaches we identify 6,499 novel N-terminal peptides with N-terminal TMTpro labelling allowing the identification of cleavage events modulated in the context of Dextran Sulfate Sodium (DSS) induced colitis within wild type and legumain-deficient colons. Among these N-termini, we identify 35 putative legumain substrates in naïve and 41 in the DSS colons supporting legumain’s role in both pro-inflammatory and physiological conditions. Use of an additional negative selection method, High-efficiency Undecanal-based N-Termini EnRichment (HUNTER), further validated and supplemented this list of identified legumain substrates. Combined, this study identifies multiple putative substrates of legumain in healthy and inflamed murine colons as well as the utility of using complementary fractionation approaches for degradomics studies.

2025-09-29 | PXD060854 | Pride

FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen

Project description:Legumain is cysteine protease primarily localised to the endo-lysosomal system. Upregulated legumain activity is associated with inflammation, neurodegeneration, and tumorigenesis. Whilst inhibiting legumain in mouse models has demonstrated therapeutic benefit, the proteolytic mechanisms underpinning its various functions are not well known and thus, further characterisation of its physiological substrates is required. Here, we developed FAIMS-enabled N-terminomics for sensitive and streamlined identification of both protein abundance changes and N-termini in complex samples. Comparison of wildtype and legumain-deficient murine spleens identified 6,366 proteins and 2,528 N-termini, of which 235 were enriched in wildtype spleens. These included 119 with asparaginyl cleavages corresponding to 110 proteins, indicating legumain-specific activity. Surprisingly, many of these localised to the nucleus and cytoplasm, hinting at novel extra-lysosomal roles of legumain. We further confirmed the direct processing of selected substrates by legumain in vitro; together, validating FAIMS-enabled N-terminomics for protease substrate detection in an unbiased and systematic manner.

2024-07-12 | PXD043122 | Pride

FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen

2024-05-23 | PXD047733 | Pride

FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen

2024-05-23 | PXD047734 | Pride

Naive and DSS Murine Colon TMTpro bRP fractionation N-terminomics

2025-09-29 | PXD060852 | Pride

TAILS N-terminomics of the human dental pulp stroma and the odontoblast layer

Project description:We report the targeted analysis of the human dental pulp stroma and the odontoblast layer using the positional proteomics technique TAILS N-terminomics, which allowed us to capture both naturally blocked and unblocked protein N-termini, and to identify differences between e.g. the dental pulp proteomes of older and younger donors. Noteworthy, these differences were most pronounced in the odontoblast region. Note: This study is a follow-up on PXD002264.

2017-10-20 | PXD006557 | Pride

Systematic optimization of C-TAILS approach for deep screening of C-terminomics

Project description:It is well known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structure and understanding protein functions. Although many efforts have been made in the fields during the latest two decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we reported an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in E. Coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to amino acid composition, more reproducible and with higher MASCOT scores. Moreover, the introduction of SCII (Single-Charge Ion Inclusion) method to alleviate the charge deficiency of small peptides allowed an additional 26 % increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. Coli in a triplicate experiments using 80 μg each. Our optimized method would benefit the deep screening of C-terminomics and possibly help discover some novel C-terminal modifications.

2015-09-15 | PXD002409 | Pride

The Identification of Proteolytic Substrates of Calpain-5 with N-terminomics

Project description:Calpain-5/CAPN5 is a calcium-activated, non-lysosomal cysteine (thiol) protease. The substrate spectrum repertoire of CAPN5 is not known. Calpains catalyze limited proteolysis of their substrates, generating novel neo protein N-termini that correspond to internal residues of their nascent substrate proteins. To identify such neo-N-termini generated by CAPN5, we employed an N-terminomics approached called the TAILS (Terminal amine isotopic labeling of substrates) proteomics method to quantitatively compare the N-terminal peptides detected in parental and CAPN5-deficient SH-SY5Y neuroblastoma cells. Thirty neo-N-termini corresponding to 29 protein groups and 24 unique proteins were found detected to be depleted in the CAPN5-/- cells. A subset of the identified putative substrates was further studied with CAPN5 co-immunoprecipitation, in vitro calcium-induced CAPN5 proteolysis assay, and comparing their cellular fragmentation patterns in parental and CAPN5-deficient SH-SY5Y cells. Here, wWe provide evidence for CAPN5-mediated proteolysis of the synaptic proteins DLGAP4, IQSEC1 and MPDZ, the neurodegeneration-related EWS, hnRNPU, TFG and UGP2, the DNA replication regulator MCM3, and the neuronal differentiation regulator LMTK1. Our data provide newThe relevance of the newly identified CAPN5 substrates tofor Neovascular neovascular inflammatory vitreoretinopathy (NIV), a progressive eye disease caused by pathogenic mutations in CAPN5, is discussed.

2025-07-21 | PXD064313 | Pride

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