Biology subjects

Makarov, A. A.

Publications and source records attributed to Makarov, A. A..

14 recordsLinked to original sources

bioRxiv2026

Orbitrap Collision Cross Section Measurements Enhance Isomer Annotations in Lipidomics

Despite advances in high-resolution mass spectrometry (HRMS), confident lipid annotation remains challenging due to the extensive chemical diversity of the lipidome and the prevalence of isomeric species. Ion mobility collision cross section (CCS) measurements provide structural information that complements HRMS; however, not all HRMS platforms can perform these measurements, necessitating a trade-off among mass resolution, accuracy, and robustness. Here, we introduce a method to infer lipid CCS values directly from liquid chromatography (LC)-Orbitrap MS experiments (OrbiCCS). We show that Orbitrap mass analyzer pressure readings, and therefore CCS values, are influenced by the LC gradient solvent composition, requiring correction using isotopically labeled internal standards injected post-column. We also show that hundreds of lipid features can be assigned OrbiCCS values in a single LC run, with average precision better than 1% and an accuracy of 1-2% relative to reference DTCCS and TIMSCCS values. This excellent CCS accuracy not only enables more reliable annotation of lipid species in complex mixtures by matching OrbiCCS values to reference databases but also accelerates lipid structural elucidation based on the unknowns position in OrbiCCS-retention time-m/z space.

biochemistry↗

bioRxiv2025

BCR repertoire analysis and cloning of antibody candidates targeting native and Asp7-isomerized beta-amyloid

Computational approaches are increasingly used to predict monoclonal antibody (mAb) candidates from BCR-seq datasets. However, the reliable identification of B cells encoding antibodies against rare antigenic epitopes remains challenging. We employed a repertoire-guided workflow combining antigen-tetramer sorting of B cells from immunized mice, followed by low-input bulk BCR-seq yielding informative clonal repertoires. Clustering and supporting somatic hypermutation (SHM) lineage analysis allowed us to identify IGH and IGK clonotypes potentially targeting {beta}-amyloid and its isoAsp7 variant (isoD7-A{beta}1-16), implicated in Alzheimers disease. We observed recurrent IGHV8-12 and IGKV1-117 usage, consistent with canonical mouse anti-A{beta} responses. Focusing on isoD7-A{beta} binders, we selected ten candidate IGH-IGK pairs for recombinant expression. One recombinant mAb demonstrated preferential binding to isoD7-A{beta} by microscale thermophoresis, supporting the feasibility of the approach but underscoring the challenge of accurate chain pairing. This highlights the potential of bioinformatic workflows to identify mAbs even under low-input conditions.

immunology↗

bioRxiv2025

Alteration in the intracellular Na+/K+ ratio regulates gene expression through the shift of G-quadruplex dynamics in living cells

Alteration in the intracellular Na+i/K+i ratio can play an important regulatory role in many mammalian cells. Here, we demonstrated that the expression of some early response genes does depend on the intracellular Na+i/K+i ratio and that the inhibition of DNA-binding activity of transcription factors c-Fos and c-Jun attenuates the upregulation of Zfp36 and Egr1 genes in HeLa cells. Both replacement of intracellular Na+ and K+ by Li+ and the dissipation of Na+i/K+i gradient demonstrated a similar effect on the transcription of Fos, Jun, Zfp36, and Ptgs2. Oligonucleotide sequences from the Fos, Kit, and My[c] promoters with an ability to fold into G-quadruplexes (G4s) change their conformations differently depending on the ratio of Na+, K+, and Li+ ions in vitro. Fluorescence lifetime imaging microscopy studies using DNA G4-specific dye DAOTA-M2 revealed that changes in compositions and ratios of monovalent metal cations in HeLa cells were accompanied by alterations in intranuclear G4s dynamics. The data demonstrate that these structures may be considered as intracellular sensors of monovalent metal cations regulating gene expression through the shift of G4s dynamics.

biochemistry↗

bioRxiv2024

Quantitative Label-Free Single-Cell Proteomics on the Orbitrap Astral MS

Single-cell proteomics by mass spectrometry (scp-MS) holds the potential to provide unprecedented insights into molecular features directly linked to the cellular phenotype, while deconvoluting complex organisms into their basic building blocks. Tailored sample preparation that maximizes the extracted amount of material that is introduced into the mass spectrometer has rapidly propelled the field forward. However, the measured signal is still at the lower edge of detection approaching the sensitivity boundary of current instrumentation. Here, we investigate the capacity of the enhanced sensitivity of the Orbitrap Astral mass spectrometer to facilitate deeper proteome profiles from low-input to single-cell samples. We carry out a comprehensive data acquisition method survey to pinpoint which parameters provide most sensitivity. Furthermore, we explore the quantitative accuracy of the obtained measurements to ensure that the obtained abundances are in line with expected ground truth values. We culminate our technical exploration by generating small datasets from two cultured cell lines and a primary bone marrow sample, to showcase obtainable proteome coverage differences from different source materials. Finally, as a proof of concept we explore protein covariation to showcase how information on known protein complexes is captured inherently in our scp-MS data.

biochemistry↗

bioRxiv2024

Narrow window data-independent acquisition on the Orbitrap Astral Mass Spectrometer enables fast and deep coverage of the plasma glycoproteome

Recently, a conceptually new mass analyzer was introduced by pairing a quadrupole Orbitrap mass spectrometer with an asymmetric track lossless (Astral) analyzer. This system provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. This instrument allows a narrow-window data-independent (nDIA) strategy, improving sensitivity and reproducibility even when using very short LC gradients. Although this represents a new technical milestone in peptide-centric proteomics, this new system has not yet been evaluated for the analyses of very complex and clinically important proteomes, such as represented by the plasma glycoproteome. Here, we evaluated the Orbitrap Astral mass spectrometer for the analysis of the plasma glycoproteome, and pioneer a dedicated nDIA workflow, themed nGlycoDIA. With substantially adjusted parameters and varying collision energies, nGlycoDIA has clear benefits for plasma glycoproteomics. We tested our method both in glycopeptide enriched and crude plasma, leading to the identification of more than 3000 unique glycoPSMs from 181 glycoproteins, covering a dynamic range of 7 orders of magnitude in the enriched plasma sample in just 40 minutes. In addition, we detect for the first time several glycosylated cytokines that have a reported plasma concentration in the ng/L range. Furthermore, shortening the gradient to 10 min still allows the detection of almost 2500 unique glycoPSMs from enriched plasma, indicating that high-throughput indepth clinical plasma glycoproteomics may be within reach.

biochemistry↗

bioRxiv2024

To 200,000 m/z and beyond: native electron capture charge reduction mass spectrometry deconvolves heterogeneous signals in large biopharmaceutical analytes

Great progress has been made in the detection of large biomolecular analytes by native mass spectrometry, however characterizing highly heterogeneous samples remains challenging due to the presence of many overlapping signals from complex ion distributions. Electron-capture charge reduction (ECCR), in which a protein cation captures free electrons without apparent dissociation, can dealign overlapping signals by shifting the ions to lower charge states. The concomitant shift to higher m/z also facilitates the exploration of instrument upper m/z limits if large complexes are used. Here we perform native ECCR on the bacterial chaperonin GroEL and megadalton scale adeno-associated virus (AAV) capsid assemblies on the Q Exactive UHMR mass spectrometer. Charge reduction of AAV8 capsids by up to 90% pushes signals well above 100,000 m/z and enables charge state resolution and mean mass determination of these highly heterogeneous samples, even for capsids loaded with genetic cargo. With minor instrument modifications the UHMR can detect charge reduced ion signals beyond 200,000 m/z. This work demonstrates the utility of ECCR for deconvolving heterogeneous signals in native mass spectrometry and presents the highest m/z signals ever recorded on an Orbitrap instrument, opening up the use of Orbitrap native mass spectrometry for heavier analytes than ever before.

molecular biology↗

bioRxiv2023

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar

Native mass spectrometry (native MS) is a powerful technique that provides information on stoichiometry, interactions, homogeneity and shape of protein complexes. However, the extent of deviation between protein structures in the mass spectrometer and in solution remains a matter of debate. Here, we uncover the gas-phase structure of {beta}-galactosidase using single particle electron cryomicroscopy (cryo-EM) down to 2.6 [A] resolution, enabled by soft-landing of mass-selected protein complexes onto cold TEM grids and in-situ ice coating. We find that large parts of the secondary and tertiary structure are retained from solution, with dehydration-driven subunit reorientation leading to consistent compaction in the gas phase. Our work enables visualizing the structure of gas-phase protein com-plexes from numerous experimental scenarios at side-chain resolution and demonstrates the possibility of more controlled cryo-EM sample preparation. One Sentence SummaryElectrospray ion-beam deposition on cold grids and in-vacuum ice growth enable cryo-EM of mass-selected proteins at 2.6 [A].

biochemistry↗

bioRxiv2023

Narrow-window DIA: Ultra-fast quantitative analysis of comprehensive proteomes with high sequencing depth

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here, we present an ultra-fast scanning data-independent acquisition (DIA) strategy consisting on 2-Th precursor isolation windows, dissolving the differences between data-dependent and independent methods. This is achieved by pairing a Quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer that provides >200 Hz MS/MS scanning speed, high resolving power and sensitivity, as well as low ppm-mass accuracy. Narrow-window DIA enables profiling of up to 100 full yeast proteomes per day, or [~]10,000 human proteins in half-an-hour. Moreover, multi-shot acquisition of fractionated samples allows comprehensive coverage of human proteomes in [~]3h, showing comparable depth to next-generation RNA sequencing and with 10x higher throughput compared to current state-of-the-art MS. High quantitative precision and accuracy is demonstrated with high peptide coverage in a 3-species proteome mixture, quantifying 14,000+ proteins in a single run in half-an-hour. TeaserAccurate and precise label-free quantification with comprehensive proteome coverage using narrow-window DIA

systems biology↗

bioRxiv2023

Characterisation of an Omnitrap-Orbitrap platform equipped with IRMPD, UVPD and ExD for the analysis of peptides and proteins

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions, that can be used to perform resonant based collision induced dissociation, several forms of electron associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation on a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the interrogation of polypeptides.

systems biology↗

bioRxiv2022

Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging when analyzing tiny sample amounts. We present a novel hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem MS scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmarked hybrid-DIA against state-of-the-art targeted MS methods (i.e. SureQuant) using EGF-stimulated HeLa cells and found the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiled the global phosphoproteome. To demonstrate the robustness, sensitivity and potential of hybrid-DIA, we profiled chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluated the difference of cancer cells in 2D vs 3D culture. Altogether, we showed that hybrid-DIA is the way-to-go method in highly sensitive phospho-proteomics experiments.

systems biology↗

bioRxiv2021

Abnormal (hydroxy)prolines deuterium content redefines hydrogen chemical mass

Analysing the {delta}2H in individual amino acids of proteins extracted from vertebrates, we unexpectedly found in some samples, notably bone collagen from seals, more than twice as much deuterium in proline and hydroxyproline residues than in seawater. This corresponds to at least four times higher {delta}2H than in any previously reported biogenic sample. We ruled out diet as a plausible mechanism for such anomalous enrichment. This finding puts into question the old adage that you are what you eat. SUMMARYThe chemical mass of hydrogen is defined as an interval from the lowest to the highest content of deuterium 2H, hydrogens heavy stable isotope. Measurements of the deviations {delta}2H in the deuterium content from the standard (ocean water, {delta}2H = 0{per thousand}) are used to characterise biological samples, such as animal bone collagen. The results are often interpreted in terms of the trophic level and diet of the animal as well as prevailing climate during its lifetime. The majority of the published bone collagen {delta}2H data fall into a narrow {delta}2H range limited to {+/-}100{per thousand}. Using novel analysis method, we unexpectedly found greatly higher {delta}2H values, up to 1500{per thousand}, in seal bone collagen. Such anomalous deuterium enrichment is detected only in two amino acid residues, proline and its derivative hydroxyproline, while other residues show much smaller {delta}2H values. Anomalously high {delta}2H values, albeit of lower magnitudes, are also found for these residues in other biological sources. This finding substantially expands the upper bound of the hydrogen chemical mass for biogenic sources. Since neither diet nor environment explain these mysteriously high enrichment levels amounting to more than twice deuterium content in sea water, our understanding of stable isotopes in nature, as well as the old adage "you are what you eat", are put in question.

biochemistry↗

bioRxiv2021

Frequency chasing of individual megadalton ions in an Orbitrap analyzer improves precision of analysis in single molecule mass spectrometry

To enhance the performance of charge detection mass spectrometry, we investigated the behavior of macromolecular single ions on their paths towards and within the Orbitrap analyzer. We discovered that ions in mass beyond one megadalton reach a plateau of stability and can be successfully trapped for seconds, travelling a path length of multiple kilometers, thereby enabling precise mass analysis with an effective resolution of greater than 100,000 at m/z 35,000. Through monitoring the frequency of individual ions, we show that these high mass ions, rather than being lost from the trap, can gradually lose residual solvent molecules and, in rare cases, a single elementary charge. Our observations highlight the importance of efficient desolvation for optimal charge detection mass spectrometry and inspired us to implement multiple improved data acquisition strategies. We demonstrate that the frequency drift of single ions due to desolvation and charge stripping can be corrected, which improves the effective ion sampling 23-fold and gives a two-fold improvement in mass precision and resolution, as demonstrated in the analysis of various viral particles.

biophysics↗

bioRxiv2019

Multiplexed Single Ion Mass Spectrometry Improves Measurement of Proteoforms and Their Complexes

A new Orbitrap-based single ion analysis procedure is shown to be possible by determining the direct charge on numerous measurements of individual protein ions to generate true mass spectra. The deployment of an Orbitrap system for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by traditional measurement of an ensemble of ions.

biochemistry↗

bioRxiv2018

pKa measurements for the SAMPL6 prediction challenge for a set of kinase inhibitor-like fragments

Determining the net charge and protonation states populated by a small molecule in an environment of interest or the cost of altering those protonation states upon transfer to another environment is a prerequisite for predicting its physicochemical and pharmaceutical properties. The environment of interest can be aqueous, an organic solvent, a protein binding site, or a lipid bilayer. Predicting the protonation state of a small molecule is essential to predicting its interactions with biological macromolecules using computational models. Incorrectly modeling the dominant protonation state, shifts in dominant protonation state, or the population of significant mixtures of protonation states can lead to large modeling errors that degrade the accuracy of physical modeling. Low accuracy hinders the use of physical modeling approaches for molecular design. For small molecules, the acid dissociation constant (pKa) is the primary quantity needed to determine the ionic states populated by a molecule in an aqueous solution at a given pH. As a part of SAMPL6 community challenge, we organized a blind pKa prediction component to assess the accuracy with which contemporary pKa prediction methods can predict this quantity, with the ultimate aim of assessing the expected impact on modeling errors this would induce. While a multitude of approaches for predicting pKa values currently exist, predicting the pKas of drug-like molecules can be difficult due to challenging properties such as multiple titratable sites, heterocycles, and tautomerization. For this challenge, we focused on set of 24 small molecules selected to resemble selective kinase inhibitors--an important class of therapeutics replete with titratable moieties. Using a Sirius T3 instrument that performs automated acid-base titrations, we used UV absorbance-based pKa measurements to construct a high-quality experimental reference dataset of macroscopic pKas for the evaluation of computational pKa prediction methodologies that was utilized in the SAMPL6 pKa challenge. For several compounds in which the microscopic protonation states associated with macroscopic pKas were ambiguous, we performed follow-up NMR experiments to disambiguate the microstates involved in the transition. This dataset provides a useful standard benchmark dataset for the evaluation of pKa prediction methodologies on kinase inhibitor-like compounds.\n\nAbbreviations

biophysics↗