Biology subjects

Oliinyk, D.

Publications and source records attributed to Oliinyk, D..

5 recordsLinked to original sources

bioRxiv2024

EXCRETE enables deep proteomics of the cyanobacterial extracellular environment

Extracellular proteins play a significant role in shaping microbial communities which, in turn, can impact ecosystem function, human health, and biotechnological processes. Yet, for many ubiquitous microbes, there is limited knowledge regarding the identity and function of secreted proteins. Here, we introduce EXCRETE (enhanced exoproteome characterization by mass spectrometry), a workflow that enables comprehensive description of microbial exoproteomes from minimal starting material. Using cyanobacteria as a case study, we benchmark EXCRETE and show a significant increase over current methods in the identification of extracellular proteins. Subsequently, we show that EXCRETE can be miniaturized and adapted to a 96-well high-throughput format. Application of EXCRETE to cyanobacteria from different habitats (Synechocystis sp. PCC 6803, Synechococcus sp. PCC 11901, and Nostoc punctiforme PCC 73102), and in different cultivation conditions, identified up to 85% of all predicted secreted proteins. Finally, functional analysis reveals that cell envelope maintenance and nutrient acquisition are central functions of the cyanobacterial secretome. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across freshwater, marine, and terrestrial species.

biochemistry↗

bioRxiv2023

μPhos: a scalable and sensitive platform for functional phosphoproteomics

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the identification and quantification of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to internal or external perturbations in space and time is crucial for further illuminating functionality of the phosphoproteome. Here we describe {micro}Phos, an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture experiments in < 8 hours total processing time. By minimizing transfer steps and reducing liquid volumes to < 200 {micro}L, we demonstrate increased sensitivity, over 90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify more than 20,000 unique phosphopeptides in a human cancer cell line using 20 {micro}g starting material, and confidently localize > 5,000 phosphorylation sites from 5 {micro}g. This depth covers key intracellular signaling pathways, rendering sample-limited applications and extensive perturbation experiments with hundreds of samples viable. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/535617v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@43d3e9org.highwire.dtl.DTLVardef@10d975forg.highwire.dtl.DTLVardef@14ce090org.highwire.dtl.DTLVardef@2b2e67_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2022

Peptide collision cross sections of 22 post-translational modifications

Recent advances have rekindled the interest in ion mobility spectrometry as an additional dimension of separation in mass spectrometry (MS)-based proteomics. It separates ions according to their size and shape in the gas phase. Here, we set out to investigate the effect of 22 different post-translational modifications (PTMs) on the collision cross section (CCS) of peptides. In total, we analyzed [~]4700 pairs of matching modified and unmodified peptide ions by trapped ion mobility spectrometry (TIMS). Linear alignment based on spike-in reference peptides resulted in highly reproducible CCS values with a median coefficient of variation of 0.3%. On a global level, we observed a redistribution in the m/z vs. ion mobility space for modified peptides upon changes in their charge state. Pairwise comparison between modified and unmodified peptides of the same charge state revealed median shifts in CCS between - 1.1% (lysine formylation) and +4.5% (O-GlcNAcylation). In general, increasing modified peptide masses were correlated with higher CCS values, in particular within homologous PTM series. However, investigating the ion populations in more detail, we found that the change in CCS can vary substantially for a given PTM depending on the gas phase structure of its unmodified counterpart. In conclusion, our study shows PTM- and sequence-specific effects on the cross section of peptides, which could be further leveraged for proteome-wide PTM analysis.

systems biology↗

bioRxiv2022

Ion mobility-resolved phosphoproteomics with dia-PASEF and short gradients

Mass spectrometry-based phosphoproteomics has identified >150,000 post-translational phosphorylation sites in the human proteome. To disentangle their functional relevance, complex experimental designs that require increased throughput are now coming into focus. Here, we apply dia-PASEF on a trapped ion mobility (TIMS) mass spectrometer to analyze the phosphoproteome of a human cancer cell line in short liquid chromatography gradients. At low sample amounts equivalent to [~]20 ug protein digest per analysis, we quantified over 12,000 phosphopeptides including [~]8,000 class I phosphosites in one hour without a spectral library. Decreasing the gradient time to 15 min yielded virtually identical coverage of the phosphoproteome, and with 7 min gradients we still quantified about 80% of the class I sites with a median coefficient of variation <10% in quadruplicates. We attribute this in part to the increased peak capacity, which effectively compensates for the higher peptide density per time unit in shorter gradients. Our data shows a five-fold reduction in the number of co-isolated peptides with TIMS. In the most extreme case, these were positional isomers of nearby phosphosites that remained unresolved with fast chromatography. In summary, we demonstrate how key features of dia-PASEF translate to phosphoproteomics, resulting in high throughput and sensitivity.

systems biology↗

bioRxiv2021

PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

Although PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

immunology↗