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SCX, 18O-labeling and charge reversal for C-terminomics

ABSTRACT: The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yields in labeling strategies or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC-MS/MS analysis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD013486 | Pride | 2019-06-20

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	1801129_Yeast_18O_Repl_3_SCX3.raw	Raw
	180324_Yeast_18O_Repl_1_SCX1.raw	Raw
	180324_Yeast_18O_Repl_1_SCX1_DMEDA.raw	Raw
	180324_Yeast_18O_Repl_1_SCX2.raw	Raw
	180324_Yeast_18O_Repl_1_SCX2_DMEDA.raw	Raw

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Publications

Combination of SCX Fractionation and Charge-Reversal Derivatization Facilitates the Identification of Nontryptic Peptides in C-Terminomics.

Kaleja Patrick P Helbig Andreas O AO Tholey Andreas A

Journal of proteome research 20190625 7

The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yielding labeling strategies, or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC-MS/MS analysis. Charge-reversal improved both MS and MS/MS spectra quality of peptides carrying ...[more]

PMID: 31195796

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Hydrophobic tagging-assisted N-termini enrichment (HYTANE) for in-depth N-terminome analysis

Project description:The analysis of protein N-termini is of great importance for understanding the protein function and elucidating the proteolytic processing. Herein, we develop a negative enrichment strategy, termed as hydrophobic tagging-assisted N-termini enrichment (HYTANE) to achieve a global N-terminome analysis. The HYTANE strategy showed a high efficiency in hydrophobic tagging and C18 material-assisted depletion using bovine serum albumin (BSA) as the sample. Furthermore, this strategy was also applied to N-termini profiling from S. cerevisiaecell lysates and enabled the identification of 1096 protein N-termini, representing the largest N-terminome dataset of S. cerevisiae. The identified N-terminal peptides accounted for 99% of all identified peptides and no deficiency in acidic and histidine-containing N-terminal peptides was observed. The presented HYTANE strategy is therefore a highly selective, efficient and unbiased strategy for the large scale N-terminome analysis.

2016-08-19 | PXD004690 | Pride

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

C-terminomics E. coli - C-terminal charge-reversal derivatization and parallel use of multiple proteases facilitates identification of protein C-termini by C-terminomics

Project description:The identification of protein C-termini in complex proteomes is challenging due to the poor ionization efficiency of the carboxyl group. Amidating the negatively charged C-termini with ethanolamine (EA) has been suggested to improve detection of C-terminal peptides and allows for a directed depletion of internal peptides after proteolysis using carboxyl reactive polymers. In the present study the derivatization with N,N-dimethylethylenediamine (DMEDA) and (4-aminobutyl)guanidine (AG) leading to a positively charged C-terminus was investigated. C-terminal charge-reversed peptides showed improved coverage of b- and y-ion series in the MS/MS spectra compared to their non-charged counterparts. DMEDA-derivatized peptides resulted in many peptides with charge states of 3+ which benefited from ETD-fragmentation. This makes the charge reversal strategy particularly useful for the analysis of protein C-termini which may also be posttranslationally modified. The labeling strategy and the indirect enrichment of C-termini worked with similar efficiency for both DMEDA and EA and their applicability was demonstrated on an E. coli proteome. Utilizing two proteases and different MS/MS activation mechanisms allowed for the identification of >400 C-termini, encompassing both canonical and truncated C-termini.

2016-03-11 | PXD003342 | Pride

Human “free-N-terminome” analysis

Project description:The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the N-terminal oriented analysis of human mitochondria enriched samples by the trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP and dN-TOP approaches (Gallien et al, 2009, Bertaccini et al. 2013)). The dN-TOP strategy based on a double light/heavy TMPP labeling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis.

2015-06-02 | PXD001521 | Pride

Human “free-N-terminome” analysis

2015-06-02 | PXD001522 | Pride

Human Dental Pulp TAILS N-terminomics

Project description:We report the targeted analysis of the human dental pulp proteome and N-terminome using the positional proteomics technique TAILS, which allowed us to capture both naturally blocked and unblocked protein N-termini. Furthermore, by using a proteomically barely studied human tissue in combination with a non-shotgun proteomics approach, we were able to identify many so far missing proteins (PE level 2-4), and established together with our previously published studies on human erythrocytes (Lange et al. 2014) and platelets (Prudova et al. 2014), a general workflow suitable for the in-depth and high-throughput analysis of protein N-termini in human specimen, as envisioned by the Chromosome-centric Human Proteome Project.

2015-08-18 | PXD002264 | 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.

2024-05-23 | PXD047733 | Pride

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

2024-05-23 | PXD047734 | Pride

Root proteome of ASPR1- deficient Arabidopsis thaliana compared to wt

Project description:Seedlings lacking the atypical aspartic protease encoded by the gene At2g03200 exhibited shorter primary roots and a pronounced reduction in the number of lateral roots. The protease was therefore name ATYPICAL ASPARTIC PROTEASE IN ROOTS 1 (ASPR1). This project compared the root proteome of a T-DNA insertion line lacking ASPR1 with wild type seedlings.

2019-02-25 | PXD009580 | Pride

Proteomic profiling of the proteolytic events in the secretome of the transformed phenotype of melanocyte-derived cells using Terminal Amine Isotopic Labeling of Substrates

Project description:The comprehensive profiling of the repertoire of secreted proteins from cancer cells and/or tissue samples provides information on the signaling events that take place during oncogenesis as well as on the cross-talk between normal and tumoral cells. Moreover, the analysis of post translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as, for example, proteolytic processing. In this context, we used Terminal Amine Isotopic Labeling of Substrates (TAILS) to perform a system-wide investigation on the N-terminome of the secretomes derived from a paired set of mouse melanocyte cell lines: Melan-a (a normal melanocyte) and Tm1 (its transformed phenotype). TAILS analysis allowed the profiling of co-translational modifications such as acetylated N-termini as well as proteolytic events in both secretomes. Although no significant difference has been found in the proportion of acetylated natural N-termini in both cell line secretomes, when evaluating amino acid identities at the scissile bond in internal peptides it was possible to detect significant differences, suggesting distinct proteolytic processes acting in the normal and tumoral secretomes. The mapping and annotation of cleavage sites in the tumoral secretome suggested functional roles of active proteases in central biological processes related to oncogenesis, such as the processing of growth factors, cleavage of extracellular matrix proteins and the shedding of ectopic domains from the cell surface, some of which may represent novel processed forms of the corresponding proteins. In the context of the tumor microenvironment, these results suggest important biological roles of proteolytic processing in murine melanoma secreted proteins.

2018-10-02 | PXD010169 | Pride

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