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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 MS1 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.
Assuntos
Proteoma , Proteômica , Espectrometria de Massas em Tandem , Humanos , Proteoma/análise , Proteômica/métodos , Fatores de TempoRESUMO
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.
Assuntos
Peptídeos , Proteômica , Proteômica/métodos , Espectrometria de Massas/métodos , Proteoma/metabolismo , Proteínas SanguíneasRESUMO
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.
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Measured angular distributions of photoelectrons from size-selected copper and sodium cluster anions are demonstrated to exhibit a universal behavior independent of the initial electron state, cluster size, or material, which can be traced back to momentum conservation upon photoemission. Quantum simulations reproduce the universality under the assumption that multielectron dynamics localizes the emission on the cluster surface and renders the cluster opaque to photoelectrons, thereby quenching interference effects that would otherwise obscure this almost classical behavior.
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We present a photoelectron imaging study of the small sodium cluster anions Na3(-), Na5(-), and Na7(-) at photon energies in the visible and near UV range (hv = 1.64-4.28 eV). The resulting angular distributions are remarkably diverse and exhibit a strong dependence on photon energy; only for hv > 3.5 eV do they evolve into more uniform distributions peaked in the direction of the laser polarization. We show that different energy dependencies of the distributions are related to different angular-momentum characters of the bound states. The jellium s-like character of the lowest single-particle states results in photoelectron emission parallel to the laser polarization at all photon energies, whereas the p-like character of the higher states leads to essentially isotropic distributions at threshold and a strong variation with photon energy. Close to the detachment threshold, the asymptotic angular distributions are attributed to the approximate validity of Wigner's law, which states that the spectrum is dominated by the partial wave with the smallest angular momentum. For the planar cluster Na5(-), we observe characteristically different behavior for electrons detached from the two in-plane p-like states, and we show how this correlates with the molecular symmetry. Our results indicate that a simple jellium-like description of the molecular orbitals is appropriate for the three-dimensional cluster Na7(-), despite the energetic splitting of the normally triply degenerate 1p level.
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Extending the fully quantum-state-resolved description of elementary chemical reactions beyond three or four atom systems is a crucial issue in fundamental chemical research. Reactions of methane with F, Cl, H or O are key examples that have been studied prominently. In particular, reactive resonances and nonintuitive mode-selective chemistry have been reported in experimental studies for the F+CH4 âHF+CH3 reaction. By investigating this reaction using transition-state spectroscopy, this joint theoretical and experimental study provides a clear picture of resonances in the F+CH4 system. This picture is deduced from high-resolution slow electron velocity-map imaging (SEVI) spectra and accurate full-dimensional (12D) quantum dynamics simulations in the picosecond regime.
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Ion traps are routinely directly coupled to mass analyzers, where they serve to suitably cool and shape an ion population prior to pulsed extraction into the analyzer proper. Such devices benefit from high duty cycle and transmission but suffer slow ion processing times caused by a compromise in the buffer gas pressure range that suitably dampens the ion kinetic energy without causing excessive scatter during extraction or within the analyzer. A rectilinear RF quadrupole ion trap has been characterized, conjoining a pressurized collision region with a pumped extraction region, and an unbroken RF interface for seamless ion transfer between them. Auxiliary electrodes mounted between the RF electrodes provide DC voltage gradients that serve to both guide ions through the device and position them at the extraction slot. The influence of the auxiliary DC upon the trapping RF field was measured, and suitable parameters were defined. A mode of operation was developed that allowed parallel processing of ions in both regions, enabling a repetition rate of 200 Hz when the device was coupled to a high-resolution accurate-mass analyzer.
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Space charge effects are the Achilles' heel of all high-resolution ion optical devices. In time-of-flight mass analyzers, these may manifest as reduction of resolving power, mass measurement shift, peak coalescence, and/or transmission losses, while highly sensitive modern ion sources and injection devices ensure that such limits are easily exceeded. Space charge effects have been investigated, by experiment and simulation study, for the astral multi-reflection analyzer, incorporating ion focusing via a pair of converging ion mirrors, and fed by a pulsed extraction ion trap. Major factors were identified as the resonant effect between ~103 ions of similar m/z in-flight and the expansion of trapped packets of ~104-5 ions prior to extraction. Optimum operation and compensated ion mirror calibration strategies were then generated and described based on these findings.
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Reflectron-based time-of-flight analyzers rely on subnanosecond detector time response to achieve acceptable resolving power for low-mid-mass, multiple-ion peaks. With the adoption of multireflection analyzers, order of magnitude longer folded ion paths relax restrictions on detector response time, allowing implementation of new technologies that greatly improve dynamic range, detector lifetime, and ion detection efficiency. A detection system is presented, integrated into a multireflection analyzer, that combines 10 keV postacceleration and focal plane correction with a unique BxE focusing, optically coupled detector, preamplification, and dual-channel digitization. Calibration and peak-handling methods are also described. The instrument demonstrated >1 × 104 dynamic range in a single shot, > 100k resolving power, and a relative immunity to detector aging.
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Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th 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 which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.
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We report high-resolution anion photoelectron spectra of thiozonide (S3(-)) acquired by slow electron velocity-map imaging (SEVI). The ions were cryogenically cooled within an ion trap before photodetachment. We measure an electron affinity of 2.3630(9) eV, resolving discrepancies in previously reported photoelectron spectra that resulted from the presence of vibrational hot bands. The SEVI spectrum shows well-resolved, extended vibrational progressions in the symmetric stretch and bending modes of S3, yielding accurate frequencies for both.
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The structure and stability of mass-selected bisulfate, sulfuric acid, and water cluster anions, HSO4(-)(H2SO4)m(H2O)n, are studied by infrared photodissociation spectroscopy aided by electronic structure calculations. The triply hydrogen-bound HSO4(-)(H2SO4) configuration appears as a recurring motif in the bare clusters, while incorporation of water disrupts this stable motif for clusters with m > 1. Infrared-active vibrations predominantly involving distortions of the hydrogen-bound network are notably missing from the infrared multiple-photon dissociation (IRMPD) spectra of these ions but are fully recovered by messenger-tagging the clusters with H2. A simple model is used to explain the observed "IRMPD transparency".
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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.
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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. 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 and structural 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.
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Anion slow photoelectron velocity-map imaging (SEVI) spectroscopy is a high-resolution variant of photoelectron spectroscopy used to study the electronic and geometric structure of atoms, molecules, and clusters. To benefit from the high resolution of SEVI when it is applied to molecular species, it is essential to reduce the internal temperature of the ions as much as possible. Here, we describe an experimental setup that combines a radio-frequency ion trap to store and cool ions with the high-resolution SEVI spectrometer. For C(5)(-), we demonstrate ion temperatures down to 10 ± 2 K after extraction from the trap, as measured by the relative populations of the two anion spin-orbit states. Vibrational hot bands and sequence bands are completely suppressed, and peak widths as narrow as 4 cm(-1) are seen due to cooling of the rotational degrees of freedom.
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Infrared multiple photon dissociation spectra for the smallest atmospherically relevant anions of sulfuric and nitric acid allow us to characterize structures and distinguish between clusters with a bisulfate or a nitrate core. We find that bisulfate is the main charge carrier for HSO(4)(-)·H(2)SO(4)·HNO(3) but not for NO(3)(-)·H(2)SO(4)·HNO(3). For the mixed dimer anion, we find evidence for the presence of two isomers: HSO(4)(-)·HNO(3) and NO(3)(-)·H(2)SO(4). Density functional calculations accompany the experimental results and provide support for these observations.
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A joint experimental-theoretical study has been carried out on electronic states of propadienylidene (H(2)CCC), using results from negative-ion photoelectron spectroscopy. In addition to the previously characterized X(1)A(1) electronic state, spectroscopic features are observed that belong to five additional states: the low-lying ã(3)B(1) and b(3)A(2) states, as well as two excited singlets, Ã(1)A(2) and B(1)B(1), and a higher-lying triplet, c(3)A(1). Term energies (T(0), in cm(-1)) for the excited states obtained from the data are: 10,354±11 (ã(3)B(1)); 11,950±30 (b(3)A(2)); 20,943±11 (c(3)A(1)); and 13,677±11 (Ã(1)A(2)). Strong vibronic coupling affects the Ã(1)A(2) and B(1)B(1) states as well as ã(3)B(1) and b(3)A(2) and has profound effects on the spectrum. As a result, only a weak, broadened band is observed in the energy region where the origin of the B(1)B(1) state is expected. The assignments here are supported by high-level coupled-cluster calculations and spectral simulations based on a vibronic coupling Hamiltonian. A result of astrophysical interest is that the present study supports the idea that a broad absorption band found at 5450 Å by cavity ringdown spectroscopy (and coincident with a diffuse interstellar band) is carried by the B(1)B(1) state of H(2)CCC.
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High resolution anion photodetachment spectra of the phenoxide and thiophenoxide anions were obtained with slow electron velocity-map imaging. The spectra show transitions to the X(2)B(1) neutral states of both species and to the Ã(2)B(2) state of the thiophenoxy radical. Comparison of the spectra with Franck-Condon simulations allows several gas-phase vibrations to be assigned. The adiabatic electron affinities are determined to be 2.2538(8) eV and 2.3542(6) eV for phenoxy and thiophenoxy, respectively. The term energy of the Ã(2)B(2) state of thiophenoxy is found to be 0.3719(9) eV, higher than the values reported in photodissociation experiments of thiophenol.
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Oxygen-doped sodium cluster anions Na(n)O(2) (-) with n=41-148 have been studied by low temperature photoelectron spectroscopy and density functional theory (DFT), with a particular emphasis on those sizes where a spherical electron shell closing is expected. The experimental spectra are in good agreement with the electronic density of states of the DFT lowest energy structures. The cluster structures show segregation between an ionically bonded molecular unit located at the cluster surface and a metallic part. The DFT calculations reveal that each oxygen atom removes two electrons from the metallic electron gas in order to become an O(2-) dianion. A jellium model would therefore predict the electron shell closings to be shifted up by four sodium atoms with respect to pure Na(n) (-) cluster anions. The electron shell closings for Na(n)O(2) (-) are located at n=43, 61, 93, and 139, so the expected four-atom shift is observed only for the small clusters of up to n=61, while a two-atom shift is observed for the larger clusters. The DFT calculations explain this departure from jellium model predictions in terms of a structural transition in the ionically bonded molecular unit.
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The transition state regions of the F + para-H2, F + normal-H2, F + CH4 and F + CD4 reactions have been studied by slow electron velocity-map imaging (SEVI) spectroscopy of the anionic precursor clusters para-FH2-, normal-FH2-, FCH4 and FCD4. The F + H2 results improve on previously published photoelectron spectra, resolving a narrow peak that appears in the same position in the para-FH2 and normal-FH2- spectra, and suggesting that additional theoretical treatment is necessary to fully describe and assign the experimental results. A small peak in the para-FH2- results is also identified, matching simulations of a product resonance in the v' = 3 vibrational level. SEVI spectra of the 2P3/2 bands of FCH4- and FCD4- show extended structure from transitions to the entrance valley van der Waals region and the reactant side of the F + CH4 transition state region. Much of this structure is attributed to bending or hindered rotation of the methane moiety and may be a spectroscopic signature of reactive resonances.