Journal: Biology

Article Title: Post-Translational Protein Deimination Signatures in Plasma and Plasma EVs of Reindeer ( Rangifer tarandus )

doi: 10.3390/biology10030222

Figure Lengend Snippet: Peptidylarginine deiminase (PAD) isoforms and deiminated proteins in reindeer plasma and plasma EVs. ( A ) Positive bands identified in reindeer plasma at expected size range of approximately 70–75 kDa using anti-human PAD2-, PAD3- and PAD4-specific antibodies (a pool of five plasma is represented). ( B ) In plasma EVs, PAD2 and PAD3 isozymes were not detected while a strong positive reaction for PAD4 was observed (EV pool from five animals). ( C ) F95-enriched IP fractions, representative of deiminated protein enrichment and isolated from reindeer plasma and plasma EVs, were stained with silverstaining following SDS-PAGE in 4–20% TGX gels. The protein size standard (std) is indicated on the blots and gels in kilodaltons (kDa).

Article Snippet: Total deiminated proteins were isolated from reindeer plasma and plasma EVs using the F95 pan-deimination antibody (MABN328, Merck, UK) and the Catch and Release ® v2.0 immunoprecipitation kit (Merck), according to previously described methods in a range of taxa [ , , , , ].

Techniques: Clinical Proteomics, Protein Enrichment, Isolation, Staining, SDS Page

Journal: Biology

Article Title: Post-Translational Protein Deimination Signatures in Plasma and Plasma EVs of Reindeer ( Rangifer tarandus )

doi: 10.3390/biology10030222

Figure Lengend Snippet: Deiminated proteins in plasma EVs of reindeer ( R. tarandus ), identified by F95 enrichment and liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. Proteins identified only in plasma EVs (and not in whole plasma) are highlighted in blue and with an asterix (*); uncharacterized hits with a secondary hit that was annotated are included and indicated in brackets. Protein ID, protein name, species hit with the Artiodactyla UniProt database, number of matches and total score are included in the table. For full detailed LC–MS/MS data for F95-enriched proteins, see Supplementary Table S1 .

Article Snippet: Total deiminated proteins were isolated from reindeer plasma and plasma EVs using the F95 pan-deimination antibody (MABN328, Merck, UK) and the Catch and Release ® v2.0 immunoprecipitation kit (Merck), according to previously described methods in a range of taxa [ , , , , ].

Techniques: Clinical Proteomics, Liquid Chromatography, Mass Spectrometry, Ubiquitin Proteomics

Journal: Biology

Article Title: Post-Translational Protein Deimination Signatures in Plasma and Plasma EVs of Reindeer ( Rangifer tarandus )

doi: 10.3390/biology10030222

Figure Lengend Snippet: Deiminated proteins in whole plasma of reindeer ( Rangifer tarandus ) identified by F95 enrichment and liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. Proteins identified only in whole plasma (and not in plasma EVs) are highlighted in pink and with an asterix (*); uncharacterized hits with a secondary hit that was annotated are included and indicated in brackets. Protein ID, protein name, species hit with the Artiodactyla UniProt database, number of matches and total score are included in the table. For full detailed LC–MS/MS data on F95-enriched proteins, see Supplementary Table S2 .

Article Snippet: Total deiminated proteins were isolated from reindeer plasma and plasma EVs using the F95 pan-deimination antibody (MABN328, Merck, UK) and the Catch and Release ® v2.0 immunoprecipitation kit (Merck), according to previously described methods in a range of taxa [ , , , , ].

Techniques: Clinical Proteomics, Liquid Chromatography, Mass Spectrometry, Ubiquitin Proteomics

Journal: Journal of Biophotonics

Article Title: Profiling Protein Citrullination in Extracellular Vesicles by Single‐Molecule Detection Using Direct Stochastic Optical Reconstruction Microscopy

doi: 10.1002/jbio.202500483

Figure Lengend Snippet: Distribution of citrullinated protein cargoes localised to EV subpopulations, comparing permeabilised (A–D) and non‐permeabilised (E–H) EVs, captured by TT or PS binding. The graphs show positive staining of EVs for pan‐citrullination (A,C,E,G) and CitH3 (B,D,F,H) respectively. The data is represented in stacked bar charts, showing the proportion of co‐stained EVs for pan‐EV, TT, and the citrullinated protein targets of interest (Cit), which were either PanCit or CitH3. EVs were either captured by TT (A,B,E,F) or by PS (C,D,G,H).

Article Snippet: The antibodies for pan‐citrullination (F95, Merck, Feltham, UK) and citrullinated histone H3 (CitH3, Abcam, Cambridge, UK) were conjugated to AlexaFluor‐647 in‐house (STFC, RAL, Harwell, UK) to use for co‐labelling experiments on EV chips from ONI (Oxford, UK).

Techniques: Binding Assay, Staining

Journal: Molecular Immunology

Article Title: Deiminated proteins in extracellular vesicles and serum of llama ( Lama glama )—Novel insights into camelid immunity

doi: 10.1016/j.molimm.2019.10.017

Figure Lengend Snippet: Deiminated proteins identified by F95 enrichment and LC–MS/MS in total serum of llama ( Lama glama ). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95 enriched eluate was analysed by LC–MS/MS and peak list files were submitted to in-house Mascot (Cambridge Proteomics). Peptide sequence hits scoring with Lama glama (LAMGL) are included as well as hits with other camelids (CAMFR= Camelus ferus ; CAMDR= Camelus dromedaries ; LAMGU= Lama guanicoe; VICPA= Vicugna pacos (Alpaca)). Hits with uncharacterized proteins are omitted in the list. For a full list of peptide sequences and m/z values see Supplementary Table 1. An asterix (*) indicates that the protein hit is specific to whole serum only.

Article Snippet: For isolation of total deiminated proteins from llama serum and serum derived EVs, the Catch and Release immunoprecipitation kit (Merck, UK) was used together with the F95 pan-deimination antibody (MABN328, Merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination ( ).

Techniques: Isolation, Immunoprecipitation, Sequencing, Ubiquitin Proteomics, Binding Assay

Journal: Molecular Immunology

Article Title: Deiminated proteins in extracellular vesicles and serum of llama ( Lama glama )—Novel insights into camelid immunity

doi: 10.1016/j.molimm.2019.10.017

Figure Lengend Snippet: Deiminated proteins identified by F95 enrichment and LC–MS/MS in EVs isolated from serum of llama ( Lama glama ). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody, the F95 enriched eluate was analysed by LC–MS/MS and peak list files were submitted to Mascot. Peptide sequence hits scoring with L. Lama glama (LAMGL) are presented as well as hits with other camelids (CAMFR= Camelus ferus ; CAMDR= Camelus dromedaries ; LAMGU= Lama guanicoe ). Hits with uncharacterised proteins are not listed. For a full list of peptide sequences and m/z values see Supplementary Table 2. An asterix (*) indicates that the protein hit is unique for EVs only.

Article Snippet: For isolation of total deiminated proteins from llama serum and serum derived EVs, the Catch and Release immunoprecipitation kit (Merck, UK) was used together with the F95 pan-deimination antibody (MABN328, Merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination ( ).

Techniques: Isolation, Immunoprecipitation, Sequencing