Biology subjects

Bensaddek, D.

Publications and source records attributed to Bensaddek, D..

10 recordsLinked to original sources

bioRxiv2024

Optimized Time-segmented Acquisition Expands Peptide and Protein Identification in TIMS-TOF Pro Mass Spectrometry

We introduce here a novel approach, termed time-segmented acquisition (Seg), to enhance the identification of peptides and proteins in trapped ion mobility spectrometry (TIMS)-Time of flight (TOF) mass spectrometry. Our method exploits the positive correlation between ion mobility values and liquid chromatography (LC) retention time to improve ion separation and resolution. By dividing the LC retention time into multiple segments and applying a segment-specific narrower ion mobility range within the TIMS tunnel, we achieved better separation and higher resolution of ion mobility. This resulted in a substantial increase in peptide identification. In comparison to conventional TIMS methods, which typically scan a static ion mobility range (either from 0.6 to 1.6 or from 0.85 to 1.3), the Seg method significantly enhances the identification of peptides, proteins and average sequence coverage per protein. These findings highlight the potential of the Seg method in expanding the capabilities of TIMS-TOF mass spectrometry, especially for peptide-focused analysis such as post-translational modifications and peptidomics.

biochemistry↗

bioRxiv2023

Systematic characterization of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry

We have developed a robust workflow for the identification of proline hydroxylation sites in proteins, using a combination of hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-Liquid Chromatography-Mass Spectrometry (LC-MS) together with refining and filtering parameters during data analysis. Using this approach, we have combined data from cell lines treated with either the prolyl hydroxylase (PHD) inhibitor, Roxadustat (FG-4592), or with the proteasome inhibitor MG-132, or with a DMSO control, to identify a total of 4,993 and 3,247 proline hydroxylation sites, respectively, in HEK293 and RCC4 cells. A subset of 1,954 (HEK293) and 1,253 (RCC4) non-collagen proline hydroxylation sites with high confidence were inhibited by FG-4592 treatment. Features characteristic of proline hydroxylated peptides were identified, which were consistent between the HEK293 and RCC4 datasets. The more hydrophilic HILIC fractions were enriched in peptides containing hydroxylated proline residues, which showed characteristic differences in charge and mass distribution, as compared with either unmodified, or oxidised peptides. Furthermore, we discovered that the intensity of the diagnostic hydroxyproline immonium ion was dependent upon parameters including the MS collision energy setting, parent peptide concentration and the sequence of amino acids adjacent to the modified proline. We show using synthetic peptides that a combination of retention time in LC and optimised MS parameter settings allows reliable identification of proline hydroxylation sites in peptides, even when multiple prolines residues are present. Focussing on proteins in which newly identified proline hydroxylation sites were inhibited by the pan-PHD inhibitor, FG-4592, showed enrichment for proteins involved in metabolism of RNA, mRNA splicing and cell cycle regulation, including the protein phosphatase 1 regulatory subunit, Repo-Man (CDCA2). We show that Repo-Man is hydroxylated at P604 and in the accompanying study by Druker et al. 1, we present a combination of cellular and biochemical evidence that hydroxylation of Repo-Man at P604 is important for its function in controlling mitotic progression.

biochemistry↗

bioRxiv2022

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA

Methylation of carbon-5 of cytosines (m5C) is a post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. m5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. We recently showed that m5C is required for Caenorhabditis elegans development and fertility under heat stress. To contribute to the understanding of how m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Our mass spectrometry analyses reveal unique binding proteins for each of the modifications. We validate our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5C in vitro. The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m5C and its derivatives.

biochemistry↗

bioRxiv2021

Molecular basis of UHRF1 allosteric activation for synergistic histone modification binding by PI5P

Chromatin marks are recognized by distinct binding modules many of which are embedded in multidomain proteins or complexes. How the different protein functionalities of complex chromatin modulators are regulated is often unclear. Using a combination of biochemical, biophysical and structural approaches we delineated the regulation of the H3unmodified and H3K9me binding activities of the multidomain UHRF1 protein. The phosphoinositide PI5P interacts with two distant flexible linker regions of UHRF1 in a mode that is dependent on the polar head group and the acyl part of the phospholipid. The associated conformational rearrangements stably position the H3unmodified and H3K9me3 histone recognition modules of UHRF1 for multivalent and synergistic binding of the H3 tail. Our work highlights a novel molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

biochemistry↗

bioRxiv2020

Erosion of human X chromosome inactivation alters proteome expression from genes across the X chromosome and all autosomes

X chromosome inactivation (XCI) is a dosage compensation mechanism in female mammals whereby genes from one X chromosome are repressed. Analysis of human induced pluripotent stem cell (iPSC) lines using proteomics, RNAseq and polysome profiling showed a major change in the proteome upon XCI erosion. This resulted in amplified RNA and protein expression from X-linked genes. However, increased protein expression was also detected from autosomal genes without a corresponding mRNA increase, altering the protein-RNA correlation between genes on the X chromosome and autosomes. Eroded iPSC lines display ~13% increase in cell protein content, along with increased expression of ribosomal proteins, ribosome biogenesis and translation factors. They also showed significantly increased levels of active polysomes within the eroded lines. We conclude that erosion of XCI causes a major remodelling of the proteome, with translational mechanisms affecting the expression of a much wider range of proteins and disease-linked loci than previously realised.

biochemistry↗

bioRxiv2019

Arabidopsis Proteome and the Mass Spectral Assay Library

Arabidopsis is an important model organism and the first plant with its genome sequenced. Knowledge from studying this species has either direct or indirect applications to agriculture and human health. Quantitative proteomics by data-independent acquisition (SWATH/DIA-MS) was recently developed and considered as a high-throughput targetedlike approach for accurate proteome quantitation. In this approach, a high-quality and comprehensive library is a prerequisite. Here, we generated a protein expression atlas of 10 organs of Arabidopsis and created a library consisting of 15,514 protein groups, 187,265 unique peptide sequences, and 278,278 precursors. The identified protein groups correspond to ~56.5% of the predicted proteome. Further proteogenomics analysis identified 28 novel proteins. We subsequently applied DIA-mass spectrometry using this library to quantify the effect of abscisic acid on Arabidopsis. We were able to recover 8,793 protein groups with 1,787 of them being differentially expressed which includes 65 proteins known to respond to abscisic acid stress. Mass spectrometry data are available via ProteomeXchange with identifier PXD012710 for data-dependent acquisition and PXD014032 for DIA analyses.\n\nO_TBL View this table:\norg.highwire.dtl.DTLVardef@1a3f799org.highwire.dtl.DTLVardef@1d4711dorg.highwire.dtl.DTLVardef@4e3f73org.highwire.dtl.DTLVardef@127a9a4org.highwire.dtl.DTLVardef@f254bb_HPS_FORMAT_FIGEXP M_TBL C_TBL

plant biology↗

bioRxiv2019

Direct interaction of PIWI and DEPS-1 is essential for piRNA function and condensate ultrastructure in Caenorhabditis elegans

Membraneless organelles are platforms for many aspects of RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. To address this question, we investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 revealed an interaction with the constitutive P granule protein DEPS-1. Furthermore we identified a novel motif on DEPS-1, PBS, which interacts directly with the Piwi domain of PRG-1. This protein complex forms intertwining ultrastructures to build elongated condensates in vivo. These sub-organelle ultrastructures depend on the Piwi-interacting motif of DEPS-1 and mediate piRNA function. Additionally, we identify a novel interactor of DEPS-1, EDG-1, which is required for DEPS-1 condensates to form correctly. We show that DEPS-1 is not required for piRNA biogenesis but piRNA function: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. Our study reveals how specific protein-protein interactions drive the spatial organisation and function of small RNA pathways within membraneless organelles.

cell biology↗

bioRxiv2018

Optimising multi-batch TMT analysis to mitigate inflation of missing values, false positives and diminished inter batch accuracy

Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, TMT in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We reveal a significant inflation rate of missing protein and peptide values and show that precision decreases as multiple batches are integrated. Additionally, we explore the effect of false positives using Y chromosome specific peptides as an internal control to quantify the effect of co-isolation interference, as well as primary and secondary reporter ion interference. Based on the results we suggest solutions to mitigate these effects. We show using a reference line can increase precision by normalising the quantification across batches and we propose experimental designs that minimise the effect of cross population reporter ion interference.

biochemistry↗

bioRxiv2018

The iPSC proteomic compendium

Induced pluripotent stem cell (iPSC) technology holds great potential for therapeutic and research purposes. The Human Induced Pluripotent Stem Cell Initiative (HipSci) was established to generate a panel of high-quality iPSCs, from healthy and disease cohorts, with accompanying multi-omics and phenotypic data. Here, we present a proteomic analysis of 217 HipSci iPSC lines obtained from 163 donors.\n\nThis dataset provides a comprehensive proteomic map of iPSCs, identifying >16,000 protein groups. We analyse how the expression profiles of proteins involved in cell cycle, metabolism and DNA repair contribute to key features of iPSC biology and we identify potential new regulators of the primed pluripotent state. To facilitate access, all these data have been integrated into the Encyclopedia of Proteome Dynamics (www.peptracker.com/epd), where it can be browsed interactively. Additionally, we generated an iPSC specific spectral library for DIA which we deposited in PRIDE along with the raw and processed mass-spectrometry data.

biochemistry↗

bioRxiv2018

Population-scale proteome variation in human induced pluripotent stem cells

Realising the potential of human induced pluripotent stem cell (iPSC) technology for drug discovery, disease modelling and cell therapy requires an understanding of variability across iPSC lines. While previous studies have characterized iPS cell lines genetically and transcriptionally, little is known about the variability of the iPSC proteome. Here, we present the first comprehensive proteomic iPSC dataset, analysing 202 iPSC lines derived from 151 donors. We characterise the major genetic determinants affecting proteome and transcriptome variation across iPSC lines and identify key regulatory mechanisms affecting variation in protein abundance. Our data identified >700 human iPSC protein quantitative trait loci (pQTLs). We mapped trans regulatory effects, identifying an important role for protein-protein interactions. We discovered that pQTLs show increased enrichment in disease-linked GWAS variants, compared with RNA-based eQTLs.

genomics↗