Biology subjects

Makarov, A.

Publications and source records attributed to Makarov, A..

12 recordsLinked to original sources

bioRxiv2026

High Resolution Multi-Pass Astral Analyzer Quantification Enables Highly Multiplexed 35-Plex Tandem Mass Tag Proteomics

Tandem mass tags (TMT) allow highly multiplexed and thus high-throughput, precisely quantitative proteomic analysis. Incorporation of additional deuterated reporter channels has near-doubled the multiplexation achieved with Thermo Scientific TMTpro reagents from 18 to 35-plex but requires extremely high [~]100k analyzer resolving power at m/z 128 to differentiate and quantify reporter ion channels, far beyond any single reflection time-of-flight analyzer, and exceeding the multi-reflection Thermo Scientific Astral analyzer in its standard operation. A multi-pass mode of Astral operation has been developed for the Thermo Scientific Orbitrap Astral Zoom mass spectrometer that triples the ion path to 90 m, more than doubling resolving power for a narrow m/z range. This "TMT HR mode" has been integrated into a new method of TMT proteomic analysis that splits regular MS2 analysis of labeled peptides into paired measurements comprising wide mass range scans for peptide identification, and TMT HR mode scans for reporter ion quantification. The method has been shown to accurately quantify 32-plex labeled HeLa protein lysate and provide far greater depth of analysis as state-of-the-art Orbitrap-only methods, while analysis of 11-plex labeled yeast showed no analytical depth sacrificed vs regular Orbitrap Astral TMT analysis. Further comparative measurements of a 2-cell line 35-plex sample demonstrated greater analytical depth, and similar quantitative precision, to "gold standard" Orbitrap MS3 methods.

biochemistry↗

bioRxiv2025

Evaluation of an Orbitrap Astral Zoom mass spectrometer prototype for quantitative proteomics - beyond identification lists

Mass spectrometry instrumentation continues to evolve rapidly yet quantifying these advances beyond conventional peptide and protein detections remain challenging. Here, we evaluate a modified Orbitrap Astral Zoom mass spectrometer (MS) prototype and compare its performance to the standard Orbitrap Astral MS. Across a range of acquisition methods and sample inputs, the prototype instrument outperformed the standard Orbitrap Astral MS in precursor and protein identifications, ion beam utilization, and quantitative precision. To enable meaningful cross-platform comparisons, we implemented an ion calibration framework that converts signal intensity from arbitrary units to ion per second. This benchmarking strategy showed that the prototype sampled 30% more ions per peptide than the original Orbitrap Astral MS. This increase in the ion beam utilization resulted in improved sensitivity and quantitative precision. To make these metrics broadly accessible, we added new metrics to the Skyline document grid to report the number of ions measured in a spectrum at the apex of the elution peak or the sum of ions between the peak integration boundaries. Taken together, our results demonstrate the prototype Orbitrap Astral Zoom as a high-performance platform for DIA proteomics and establish a generalizable framework for evaluation of mass spectrometer performance based on the number of ions detected for each analyte. Data are available on Panorama Public and on ProteomeXchange under the identifier PXD064536.

biochemistry↗

bioRxiv2024

Pentose Phosphate Pathway Protects E. coli from Antibiotic Lethality

Disruption of both branches of the canonical pentose phosphate pathway (PPP) in E. coli by combined inactivation of the zwf and talAB genes provokes the restoration of the ancient anabolic variant of PPP (aPPP). In the aPPP, pentose-5-phosphates are synthesized unidirectionally from fructose-6-phosphate and glyceraldehyde-3-phosphate by transketolase B, aldolase A, and phosphatase GlpX, converting sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphate. Unexpectedly, the double zwf talAB mutant exhibits decreased survival after treatment by diverse classes of antibiotics with little effect on the minimal inhibitory concentration. Simultaneously, we found that killing effect of antimicrobials on the zwf talAB mutant could be reversed by the inactivation of either purR or deoB genes, both responsible for ribose-5-phosphate content in the mutant strain. Enhanced biosynthesis of the cell wall component ADP-heptose from sedoheptulose-7-phosphate also suppressed killing effect of antibiotics on the zwf talAB mutant. Furthermore, the inactivation of the Entner-Doudoroff pathway ({Delta}edd) or shifting the metabolic equilibrium by the addition of exogenous phosphogluconate reverts aPPP to glycolysis, preventing the accumulation of excess pentose phosphates and the occurrence of the futile cycle in zwf talAB cells, thus desensitizing them to antibiotics. Our findings show that ribose-5-phosphate metabolism plays a crucial role in bacterial tolerance to a wide range of bactericidal antibiotics. We propose that targeting PPP could be a promising strategy for developing new therapeutic agents aimed at potentiating clinically significant antimicrobials. IMPORTANCERecent studies have revealed the crucial role of bacterial cells metabolic status in its susceptibility to the lethal action of antibacterial drugs. However, there is still no clear understanding of which key metabolic nodes are optimal targets to improve the effectiveness of bacterial infection treatment. Our study establishes that the disruption of the canonical pentose phosphate pathway induces one-way anabolic synthesis of pentose phosphates (aPPP) in E. coli cells, significantly increasing the killing efficiency of various antibiotics. It is also demonstrated that the activation of ribose-5-phosphate utilization processes restores bacterial tolerance to antibiotics. We consider the synthesis of ribose-5-phosphate to be one of the determining factors of bacterial cell stress resistance. Understanding bacterial metabolic pathways, particularly the aPPPs role in antibiotic sensitivity, offers insights for developing novel adjuvant therapeutic strategies to enhance antibiotic potency.

microbiology↗

bioRxiv2023

Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics

The complexity of human physiology arises from well-orchestrated interactions between trillions of single cells in the body. While single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of cell diversity, gene expression alone does not fully characterize cell phenotypes. Additional molecular dimensions, such as proteins, are needed to define cellular states accurately. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool for comprehensive protein analysis, including single-cell applications. However, challenges remain in terms of throughput and proteomic depth, in order to maximize the biological impact of single-cell proteomics by Mass Spectrometry (scp-MS) workflows. This study leverages a novel high-resolution, accurate mass (HRAM) instrument platform, consisting of both an Orbitrap and an innovative HRAM Asymmetric Track Lossless (Astral) analyzer. The Astral analyzer offers high sensitivity and resolution through lossless ion transfer and a unique flight track design. We evaluate the performance of the Thermo Scientific Orbitrap Astral MS using Data-Independent Acquisition (DIA) and assess proteome depth and quantitative precision for ultra-low input samples. Optimal DIA method parameters for single-cell proteomics are identified, and we demonstrate the ability of the instrument to study cell cycle dynamics in Human Embryonic Kidney (HEK293) cells, and cancer cell heterogeneity in a primary Acute Myeloid Leukemia (AML) culture model.

systems biology↗

bioRxiv2023

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend towards high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time, and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass resolving quadrupole, the Orbitrap and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared to state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with high dynamic range and resolution, synchronized with Astral analyzers acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/543408v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1eedfdeorg.highwire.dtl.DTLVardef@1ab7939org.highwire.dtl.DTLVardef@a3b2f8org.highwire.dtl.DTLVardef@13ac46b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Evaluating the performance of the Astral mass analyzer for quantitative proteomics using data independent acquisition

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

biochemistry↗

bioRxiv2022

Scanning ion-conductance microscopy for studying β-amyloid aggregate formation on living cell surface

Alzheimers disease (AD) is the most common form of dementia, a progressive neurological disorder characterized by short and long-term memory loss, including cognitive and functional impairment, which is refractory to current therapy. It is suggested that the aggregation of {beta}-amyloid (A{beta}) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules, and consequently - alterations of cell mechanical properties. Thus, the effect of amyloid oligomers on cells Youngs modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Youngs modulus mapping and confocal imaging of A{beta} aggregates formation on living cell surfaces with subsequent assessment of the reactive oxygen species levels inside single cells using platinum nanoelectrodes. We showed that correlative SICM technique, in conjunction with topography mapping and confocal imaging, can be used for Patch-Clamp recordings from living cells with evidently formed FAM-labeled A{beta} aggregates on its surface. As we demonstrated, SICM can be successfully applied to studying cytotoxicity mechanisms of A{beta} aggregates on living cell surface.

cell biology↗

bioRxiv2021

Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition

Electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA) have revolutionized structure determination of homogeneous proteins. However, obtaining high-resolution structures from heterogeneous samples remains a major challenge, as the various protein states embedded in thin films of vitreous ice may be classified incorrectly, resulting in detrimental averaging of features. Here we present native electrospray ion-beam deposition (native ES-IBD) for the preparation of extremely high-purity cryo-EM samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, and subsequently frozen in liquid nitrogen. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected proteins and protein assemblies. SPA reveals that they remain structurally intact, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM structure determination.

molecular biology↗

bioRxiv2021

Structural remodeling of SARS-CoV-2 spike protein glycans reveals the regulatory roles in receptor binding affinity

Glycans of the SARS-CoV-2 spike protein are speculated to play functional roles in the infection processes as they extensively cover the protein surface and are highly conserved across the variants. To date, the spike protein has become the principal target for vaccine and therapeutic development while the exact effects of its glycosylation remain elusive. Experimental reports have described the heterogeneity of the spike protein glycosylation profile. Subsequent molecular simulation studies provided a knowledge basis of the glycan functions. However, there are no studies to date on the role of discrete glycoforms on the spike protein pathobiology. Building an understanding of its role in SARS-CoV-2 is important as we continue to develop effective medicines and vaccines to combat the disease. Herein, we used designed combinations of glycoengineering enzymes to simplify and control the glycosylation profile of the spike protein receptor-binding domain (RBD). Measurements of the receptor binding affinity revealed the regulatory effects of the RBD glycans. Remarkably, opposite effects were observed from differently remodeled glycans, which presents a potential strategy for modulating the spike protein behaviors through glycoengineering. Moreover, we found that the reported anti-SARS-CoV-(2) antibody, S309, neutralizes the impact of different RBD glycoforms on the receptor binding affinity. Overall, this work reports the regulatory roles that glycosylation plays in the interaction between the viral spike protein and host receptor, providing new insights into the nature of SARS-CoV-2. Beyond this study, enzymatic remodeling of glycosylation offers the opportunity to understand the fundamental role of specific glycoforms on glycoconjugates across molecular biology. Covert art LegendsThe glycosylation of the SARS-CoV-2 spike protein receptor-binding domain has regulatory effects on the receptor binding affinity. Sialylation or not determines the "stabilizing" or "destabilizing" effect of the glycans. (Protein structure model is adapted from Protein Data Bank: 6moj. The original model does not contain the glycan structure.) SignificanceGlycans extensively cover the surface of SARS-CoV-2 spike (S) protein but the relationships between the glycan structures and the protein pathological behaviors remain elusive. Herein, we simplified and harmonized the glycan structures in the S protein receptor-binding domain and reported their regulatory roles in human receptor interaction. Opposite regulatory effects were observed and were determined by discrete glycan structures, which can be neutralized by the reported S309 antibody binding to the S protein. This report provides new insight into the mechanism of SARS-CoV-2 S protein infection as well as S309 neutralization.

biochemistry↗

bioRxiv2019

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 unique peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 minute LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

biochemistry↗

bioRxiv2019

Resolving heterogeneous high-mass macromolecular machineries by Orbitrap-based single particle charge detection mass spectrometry

Here we show that single particle charge-detection mass spectrometry (CD-MS) can be performed on a ubiquitous Orbitrap mass analyser and applied to the analysis of high-mass (megadalton) heterogeneous biomolecular assemblies. We demonstrate that single particle high-mass ions can survive in the Orbitrap for seconds, whereby their measured signal amplitudes scale linearly with charge state over the entire m/z range. Orbitrap based single particle CD-MS can be used to resolve mixed ion populations, accurately predict charge states, and consequently also the mass of the ions. We successfully applied CD-MS to challenging natural and biotherapeutic protein assemblies, such as IgM oligomers, designed protein nano-cages, ribosome particles and intact, empty- and genome-loaded Adeno-associated virus particles. Single particle CD-MS combined with native MS on existing Orbitrap platforms will greatly expand its application, especially in the mass analysis of megadalton heterogeneous biomolecular assemblies.

biophysics↗

bioRxiv2018

Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

biochemistry↗