Dataset Information

A Collision-induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-based Quantification in Proteomics

ABSTRACT: Fragment ion-based proteome quantification methods quantify peptides based on unique peptide fragment ions avoiding the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a novel approach that relies on a collision-induced dissociation (CID)-cleavable isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragment ions while reducing the complexity of the b-ion series thus facilitating data processing. Multiplex labelling is based on selective N-terminal dimethylation followed by derivatizing the C-terminal Lys residue of LysC peptides with isobaric Ac-IPG tags with different isotope distributions on Pro-Gly and Ac-Ile. Upon fragmentation between Ile and Pro, the resulting y-ions with the neutral loss of Ac-Ile can be distinguished between the different labelling channels based on different numbers of isotope labels on the Pro-Gly part. The peak intensities of y-ions contain the information for relative quantification.

INSTRUMENT(S): Q Exactive

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

SUBMITTER: Xiaobo Tian

LAB HEAD: Rainer Bischoff

PROVIDER: PXD018790 | Pride | 2020-08-17

REPOSITORIES: Pride

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Publications

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics.

Tian Xiaobo X de Vries Marcel P MP Permentier Hjalmar P HP Bischoff Rainer R

Journal of proteome research 20200817 9

Quantifying peptides based on unique peptide fragment ions avoids the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a collision-induced dissociation-cleavable, isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragments while reducing the complexity of the b-ion series compared to conventional fragment ion-based quantification methods thus facilitat ...[more]

PMID: 32786690

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Project description:Acanthamoeba castellanii (Ac) and macrophages share structural, morphological, physiological and biochemical similarities, including the ability to phagocyte a myriad of microorganisms in the environments they live. Whereas there is voluminous information on phagocytic receptors of macrophages, for Ac, the understanding of how the recognition of extracellular microorganisms works still awaits elucidation. Recently, our group described mannose-binding proteins expressed on the surface of the trophozoites. However, as soluble mannose did not inhibit entirely the process, other interactions might be possible. In the present work, we aimed to characterize the potential Ac proteins able to recognize the polysaccharide β-1,3-glucan on fungal surfaces. Our data demonstrate that Ac could bind curdlan or laminarin on its surface as detected by Dectin-1-Fc and fluorescent conjugate, suggesting the presence of β-1,3-glucan binding molecules. Optical tweezers detected higher adhesion affinity of laminarin or curdlan coated beads to A. castellanii (characteristic time of 46.9 s and 43.9 s, respectively) in comparison control beads (BSA or dextran-coated). In agreement, a H. capsulatum (Hc) G217B having β-1,3-glucan as the most external layer strongly adhered to Ac (characteristic time of 5.3 s), whereas Hc G186A, an α-1,3-glucan expressing strain, displayed much lower adhesion forces (characteristic time of 83.6 s). The specificity of our system was confirmed with addition of soluble β-1,3-glucan, which inhibited dramatically the adhesion of Hc G217B to Ac (characteristic time of 38,5 s). By indirect ELISA, the biotinylated extract of Ac showed higher binding to Hc G217B surface than Hc G186A, as similar results were observed when using Dectin-1-Fc. By interaction assays, association rates to Ac and RAW macrophages were twice higher for Hc G217B when compared to Hc G186A. Inhibitions with mannose, or its combinations with curdlan or laminarin demonstrated inhibitions higher than 50% during Ac and Hc G217B interaction. For RAW macrophages, the combinations mannose + laminarin and mannose + curdlan had inhibition of 64.4% and 51.5%, respectively, in the interaction with Hc G217B. The killing assay show that for A. castellanii, there was a decrease in the number of viable fungi when either laminarin and curdlan were added, which is similar to results observed with macrophages, suggesting the participation of this receptor for fungal entrance and survival within phagocytes. Proteomics identified several proteins with the capacity to bind β-1,3-glucans, including a membrane integral component (L8HDD6) displaying a legume lectin domain and also belonging to the Concanavalin A-like lectin/glucanase domain superfamily. By the demonstrated binding specificity of this receptor, our data reinforce other pathways of fungal recognition and suggests to a possible parallel or even divergent evolution, between A. castellanii and macrophages.

Dataset Information

A Collision-induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-based Quantification in Proteomics

Publications

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics.

Similar Datasets

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