The One Hour Human Proteome.

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.

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 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.

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

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.

High-resolution mass spectrometry of small molecules bound to membrane proteins.

Small molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry-based method for analyzing intact membrane protein-ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.

Phase-Constrained Spectrum Deconvolution for Fourier Transform Mass Spectrometry.

This Article introduces a new computationally efficient noise-tolerant signal processing method, referred to as phased spectrum deconvolution method (ΦSDM), designed for Fourier transform mass spectrometry (FT MS). ΦSDM produces interference-free mass spectra with resolution beyond the Fourier transform (FT) uncertainty limit. With a presumption that the oscillation phases are preserved, the method deconvolves an observed FT spectrum into a distribution of harmonic components bound to a fixed frequency grid, which is several times finer than that of FT. The approach shows stability under noisy conditions, and the noise levels in the resulting spectra are lower than those of the original FT spectra. Although requiring more computational power than standard FT algorithms, ΦSDM runs in a quasilinear time. The method was tested on both synthetic and experimental data, and consistently demonstrated performance superior to the FT-based methodologies, be it across the entire mass range or on a selected mass window of interest. ΦSDM promises substantial improvements in the spectral quality and the speed of FT MS instruments. It might also be beneficial for other spectroscopy approaches which require harmonic analysis for data processing.

The Q Exactive HF, a Benchtop mass spectrometer with a pre-filter, high-performance quadrupole and an ultra-high-field Orbitrap analyzer.

The quadrupole Orbitrap mass spectrometer (Q Exactive) made a powerful proteomics instrument available in a benchtop format. It significantly boosted the number of proteins analyzable per hour and has now evolved into a proteomics analysis workhorse for many laboratories. Here we describe the Q Exactive Plus and Q Exactive HF mass spectrometers, which feature several innovations in comparison to the original Q Exactive instrument. A low-resolution pre-filter has been implemented within the injection flatapole, preventing unwanted ions from entering deep into the system, and thereby increasing its robustness. A new segmented quadrupole, with higher fidelity of isolation efficiency over a wide range of isolation windows, provides an almost 2-fold improvement of transmission at narrow isolation widths. Additionally, the Q Exactive HF has a compact Orbitrap analyzer, leading to higher field strength and almost doubling the resolution at the same transient times. With its very fast isolation and fragmentation capabilities, the instrument achieves overall cycle times of 1 s for a top 15 to 20 higher energy collisional dissociation method. We demonstrate the identification of 5000 proteins in standard 90-min gradients of tryptic digests of mammalian cell lysate, an increase of over 40% for detected peptides and over 20% for detected proteins. Additionally, we tested the instrument on peptide phosphorylation enriched samples, for which an improvement of up to 60% class I sites was observed.

High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies.

The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution.

Defining the stoichiometry and cargo load of viral and bacterial nanoparticles by Orbitrap mass spectrometry.

Accurate mass analysis can provide useful information on the stoichiometry and composition of protein-based particles, such as virus-like assemblies. For applications in nanotechnology and medicine, such nanoparticles are loaded with foreign cargos, making accurate mass information essential to define the cargo load. Here, we describe modifications to an Orbitrap mass spectrometer that enable high mass analysis of several virus-like nanoparticles up to 4.5 MDa in mass. This allows the accurate determination of the composition of virus-like particles. The modified instrument is utilized to determine the cargo load of bacterial encapsulin nanoparticles that were engineered to encapsulate foreign cargo proteins. We find that encapsulin packages from 8 up to 12 cargo proteins, thereby quantifying cargo load but also showing the ensemble spread. In addition, we determined the previously unknown stoichiometry of the three different splice variants of the capsid protein in adeno-associated virus (AAV) capsids, showing that symmetry is broken and assembly is heterogeneous and stochastic. These results demonstrate the potential of high-resolution mass analysis of protein-based nanoparticles, with widespread applications in chemical biology and nanotechnology.

Discrimination of leucine and isoleucine in peptides sequencing with Orbitrap Fusion mass spectrometer.

An efficient approach to easy and reliable differentiation between isomeric leucine and isoleucine in peptide sequencing utilizes multistage electron transfer dissociation and higher energy collision activated dissociation in the Orbitrap Fusion mass spectrometer. The MS(3) method involves production and isolation of primary odd-electron z(•) ions, followed by radical site initiation of their fragmentation with formation of w-ions, characteristic of the isomeric amino acid residues. Six natural nontryptic peptides isolated from the secretion of frog Rana ridibunda were studied. Their lengths were in the range between 15 and 37 amino acids and the number of targeted isomeric (Leu/Ile) residues varied between 1 and 7. The experiments were successful in all 22 cases of Leu/Ile residues, leaving no doubts in identification. The method is extremely selective as the targeted w-ions appear to be the most intense in the spectra. The proposed approach may be incorporated into shotgun proteomics algorithms and allows for the development of an exclusively mass spectrometric method for automated complete de novo sequencing of various peptides and proteins.

Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS.

The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.

Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes.

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

Paradigm shift in biomarker translation: a pipeline to generate clinical grade biomarker candidates from DIA-MS discovery.

Clinical biomarker development has been stymied by inaccurate protein quantification from mass spectrometry (MS) discovery data and a prolonged validation process. To mitigate these issues, we created the Targeted Extraction Assessment of Quantification (TEAQ) software package. This innovative tool uses the discovery cohort analysis to select precursors, peptides, and proteins that adhere to established targeted assay criteria. TEAQ was applied to Data-Independent Acquisition MS data from plasma samples acquired on an Orbitrap™ Astral™ MS. Identified precursors were evaluated for linearity, specificity, repeatability, reproducibility, and intra-protein correlation from 11-point loading curves under three throughputs, to develop a resource for clinical-grade targeted assays. From a clinical cohort of individuals with inflammatory bowel disease (n=492), TEAQ successfully identified 1116 signature peptides for 327 quantifiable proteins from 1180 identified proteins. Embedding stringent selection criteria adaptable to targeted assay development into the analysis of discovery data will streamline the transition to validation and clinical studies.

Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.

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.

From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry.

Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS(3), from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194 kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highly charged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.

Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer.

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.

Elucidating the Mechanism of Multispecific Antibody Aggregation through Subunit Analysis.

Multispecific antibody constructs are quickly becoming more common constructs in biopharmaceuticals to improve specificity and efficacy. While the advent of this technology has led to improved therapeutics, its development has challenged the analytical tools through which these therapeutics are characterized. Moreover, new critical quality attributes, such as aggregation, have challenged the approaches to characterization even further. Herein, we describe a novel native subunit analysis using IdeS and IgdE analyzed by native size exclusion chromatography coupled with mass spectrometry to interrogate the mechanism of aggregation in a multispecific antibody. Digestion by IdeS and IdgE allows for the retention and detection of noncovalent interactions thereafter. Aggregation was localized to single-chain fragment variables (scFvs) wherein a domain swapping mechanism between VH1/VL2 and VH2/VL1 occurs.

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.

Fast and Deep Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer.

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.

Protein Coronas on Functionalized Nanoparticles Enable Quantitative and Precise Large-Scale Deep Plasma Proteomics.

Background: The wide dynamic range of circulating proteins coupled with the diversity of proteoforms present in plasma has historically impeded comprehensive and quantitative characterization of the plasma proteome at scale. Automated nanoparticle (NP) protein corona-based proteomics workflows can efficiently compress the dynamic range of protein abundances into a mass spectrometry (MS)-accessible detection range. This enhances the depth and scalability of quantitative MS-based methods, which can elucidate the molecular mechanisms of biological processes, discover new protein biomarkers, and improve comprehensiveness of MS-based diagnostics. Methods: Investigating multi-species spike-in experiments and a cohort, we investigated fold-change accuracy, linearity, precision, and statistical power for the using the Proteograph™ Product Suite, a deep plasma proteomics workflow, in conjunction with multiple MS instruments. Results: We show that NP-based workflows enable accurate identification (false discovery rate of 1%) of more than 6,000 proteins from plasma (Orbitrap Astral) and, compared to a gold standard neat plasma workflow that is limited to the detection of hundreds of plasma proteins, facilitate quantification of more proteins with accurate fold-changes, high linearity, and precision. Furthermore, we demonstrate high statistical power for the discovery of biomarkers in small- and large-scale cohorts. Conclusions: The automated NP workflow enables high-throughput, deep, and quantitative plasma proteomics investigation with sufficient power to discover new biomarker signatures with a peptide level resolution.

Exploring an orbitrap analyzer for the characterization of intact antibodies by native mass spectrometry.

Antibody profiling: native mass spectrometry analysis of intact antibodies can be achieved with improved speed, sensitivity, and mass resolution by using a modified orbitrap instrument. Complex mixtures of monoclonal antibodies can be resolved and their glycan "fingerprints" can be profiled. Noncovalent interactions are maintained, thus allowing antibody-antigen binding to be measured.