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Physiological Reviews Jul 2024Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at a molecular level. Proteomics provides... (Review)
Review
Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at a molecular level. Proteomics provides biologists with a snapshot of context-dependent protein and proteoform expression, structural conformations, dynamic turnover, and protein-protein interactions. Cardiac proteomics can offer a broader and deeper understanding of the molecular mechanisms that underscore cardiovascular disease, and it is foundational to the development of future therapeutic interventions. This review encapsulates the evolution, current technologies, and future perspectives of proteomic-based mass spectrometry as it applies to the study of the heart. Key technological advancements have allowed researchers to study proteomes at a single-cell level and employ robot-assisted automation systems for enhanced sample preparation techniques, and the increase in fidelity of the mass spectrometers has allowed for the unambiguous identification of numerous dynamic posttranslational modifications. Animal models of cardiovascular disease, ranging from early animal experiments to current sophisticated models of heart failure with preserved ejection fraction, have provided the tools to study a challenging organ in the laboratory. Further technological development will pave the way for the implementation of proteomics even closer within the clinical setting, allowing not only scientists but also patients to benefit from an understanding of protein interplay as it relates to cardiac disease physiology.
Topics: Animals; Humans; Proteomics; Cardiovascular Diseases; Heart; Protein Processing, Post-Translational; Mass Spectrometry
PubMed: 38300522
DOI: 10.1152/physrev.00026.2023 -
Current Opinion in Structural Biology Dec 2023Structure determination by single-particle cryoEM has matured into a core structural biology technique. Despite many methodological advancements, most cryoEM grids are... (Review)
Review
Structure determination by single-particle cryoEM has matured into a core structural biology technique. Despite many methodological advancements, most cryoEM grids are still prepared using the plunge-freezing method developed ∼40 years ago. Embedding samples in thin films and exposing them to the air-water interface often leads to sample damage and preferential orientation of the particles. Using native mass spectrometry to create cryoEM samples, potentially avoids these problems and allows the use of mass spectrometry sample isolation techniques during EM grid creation. We review the recent publications that have demonstrated protein complexes can be ionized, flown through the mass spectrometer, gently landed onto EM grids, imaged, and reconstructed in 3D. Although many uncertainties and challenges remain, the combination of cryoEM and MS has great potential.
Topics: Cryoelectron Microscopy; Water; Mass Spectrometry
PubMed: 37703606
DOI: 10.1016/j.sbi.2023.102699 -
Frontiers in Chemistry 2023Mass measurements in the mega-to giga-Dalton range are essential for the characterization of natural and synthetic nanoparticles, but very challenging to perform using...
Mass measurements in the mega-to giga-Dalton range are essential for the characterization of natural and synthetic nanoparticles, but very challenging to perform using conventional mass spectrometers. Nano-electro-mechanical system (NEMS) based MS has demonstrated unique capabilities for the analysis of ultra-high mass analytes. Yet, system designs to date included constraints transferred from conventional MS instruments, such as ion guides and high vacuum requirements. Encouraged by other reports, we investigated the influence of pressure on the performances of the NEMS sensor and the aerodynamic focusing lens that equipped our first-generation instrument. We thus realized that the NEMS spectrometer could operate at significantly higher pressures than anticipated without compromising particle focusing nor mass measurement quality. Based on these observations, we designed and constructed a new NEMS-MS prototype considerably more compact than our original system, and which features an improved aerodynamic lens alignment concept, yielding superior particle focusing. We evaluated this new prototype by performing nanoparticle deposition to characterize aerodynamic focusing, and mass measurements of calibrated gold nanoparticles samples. The particle capture efficiency showed nearly two orders of magnitude improvement compared to our previous prototype, while operating at two orders of magnitude greater pressure, and without compromising mass resolution.
PubMed: 37841207
DOI: 10.3389/fchem.2023.1238674 -
Molecular & Cellular Proteomics : MCP May 2024We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation,...
We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.
Topics: Humans; Proteome; Tandem Mass Spectrometry; Proteomics; Time Factors
PubMed: 38579929
DOI: 10.1016/j.mcpro.2024.100760 -
Analytical Chemistry Oct 2023The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the...
The growing trend toward 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 with 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 a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.
PubMed: 37815927
DOI: 10.1021/acs.analchem.3c02856 -
BioRxiv : the Preprint Server For... May 2024Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein...
Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology was benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.
PubMed: 38045259
DOI: 10.1101/2023.11.21.568149 -
JACS Au Aug 2023Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the...
Crosslinking mass spectrometry provides pivotal information on the structure and interaction of proteins. MS-cleavable crosslinkers are regarded as a cornerstone for the analysis of complex mixtures. Yet they fragment under similar conditions as peptides, leading to mixed fragmentation spectra of the crosslinker and peptide. This hampers selecting individual peptides for their independent identification. Here, we introduce orthogonal cleavage using ultraviolet photodissociation (UVPD) to increase crosslinker over peptide fragmentation. We designed and synthesized a crosslinker that can be cleaved at 213 nm in a commercial mass spectrometer configuration. In an analysis of crosslinked lysate, the crosslinker-to-peptide fragment intensity ratio increases from nearly 1 for a conventionally cleavable crosslinker to 5 for the UVPD-cleavable crosslinker. This largely increased the sensitivity of selecting the individual peptides for MS3, even more so with an improved doublet detection algorithm. Data are available via ProteomeXchange with identifier PXD040267.
PubMed: 37654600
DOI: 10.1021/jacsau.3c00199 -
Journal of Proteome Research Oct 2023We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific...
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 extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.
Topics: Proteomics; Mass Spectrometry; Peptides; Proteome; Blood Proteins
PubMed: 37683181
DOI: 10.1021/acs.jproteome.3c00357 -
Molecules (Basel, Switzerland) Nov 2023Although a quadruple mass analyzer and an ion trap mass analyzer have complementary analytical features, they usually have different geometries, operational modes, and...
Although a quadruple mass analyzer and an ion trap mass analyzer have complementary analytical features, they usually have different geometries, operational modes, and electronic control systems. As a continuous effort to extend its coverage, both quadrupole and ion trap operation modes were realized on a "brick" miniature mass spectrometer with a single mass analyzer. In the quadrupole operation mode, low-mass ions ranging from 31 to 502 Th can be analyzed. On the other hand, the ion trap mode can be utilized to cover ions with higher mass to charge ratios (up to 922 Th), as well as performing tandem mass spectrometry. To realize the multiplexing of both operation modes, a printed circuit board (PCB)-based multi-electrode quadrupole-ion trap mass analyzer was designed and integrated in the system. To cover both volatile and non-volatile molecules, two ionization sources were also implemented, including a nano electrospray ionization source and an in-vacuum plasma ionization source. Performances of the instrument operated in these two modes were characterized, such as mass resolution, sensitivity, and mass range. Results demonstrate that the combination of the quadrupole and ion trap operation modes can provide new capabilities when solving analytical problems.
PubMed: 38005361
DOI: 10.3390/molecules28227640