Autopilot: An Online Data Acquisition Control System for the Enhanced High-Throughput Characterization of Intact Proteins

The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On <i>Pseudomonas aeruginosa</i>, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context

Autopilot: An Online Data Acquisition Control System for the Enhanced High-Throughput Characterization of Intact Proteins

The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On <i>Pseudomonas aeruginosa</i>, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context

Fragmentation of Integral Membrane Proteins in the Gas Phase

Integral membrane proteins (IMPs) are of great biophysical and clinical interest because of the key role they play in many cellular processes. Here, a comprehensive top down study of 152 IMPs and 277 soluble proteins from human H1299 cells including 11 087 fragments obtained from collisionally activated dissociation (CAD), 6452 from higher-energy collisional dissociation (HCD), and 2981 from electron transfer dissociation (ETD) shows their great utility and complementarity for the identification and characterization of IMPs. A central finding is that ETD is ∼2-fold more likely to cleave in soluble regions than threshold fragmentation methods, whereas the reverse is observed in transmembrane domains with an observed ∼4-fold bias toward CAD and HCD. The location of charges just prior to dissociation is consistent with this directed fragmentation: protons remain localized on basic residues during ETD but easily mobilize along the backbone during collisional activation. The fragmentation driven by these protons, which is most often observed in transmembrane domains, both is of higher yield and occurs over a greater number of backbone cleavage sites. Further, while threshold dissociation events in transmembrane domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from the nearest charge site (R, K, H, N-terminus), fragmentation is strongly influenced by the N- or C-terminal position relative to that site: the ratio of observed b- to y-fragments is ∼1:3 if the cleavage occurs >7 residues N-terminal and ∼3:1 if it occurs >7 residues C-terminal to the nearest basic site. Threshold dissociation products driven by a mobilized proton appear to be strongly dependent on not only relative position of a charge site but also N- or C-terminal directionality of proton movement

'Iter Dalekarlicum'

Mass spectrometry based proteomics generally seeks to identify and fully characterize protein species with high accuracy and throughput. Recent improvements in protein separation have greatly expanded the capacity of top-down proteomics (TDP) to identify a large number of intact proteins. To date, TDP has been most tightly associated with Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Here, we couple the improved separations to a Fourier-transform instrument based not on ICR but using the Orbitrap Elite mass analyzer. Application of this platform to H1299 human lung cancer cells resulted in the unambiguous identification of 690 unique proteins and over 2000 proteoforms identified from proteins with intact masses <50 kDa. This is an early demonstration of high throughput TDP (>500 identifications) in an Orbitrap mass spectrometer and exemplifies an accessible platform for whole protein mass spectrometry

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)-MS³, 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 (<i>m</i>/<i>z</i>) 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

Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer

Over the past decade, developments in high resolution mass spectrometry have enabled the high throughput analysis of intact proteins from complex proteomes, leading to the identification of thousands of proteoforms. Several previous reports on top-down proteomics (TDP) relied on hybrid ion trap–Fourier transform mass spectrometers combined with data-dependent acquisition strategies. To further reduce TDP to practice, we use a quadrupole-Orbitrap instrument coupled with software for proteoform-dependent data acquisition to identify and characterize nearly 2000 proteoforms at a 1% false discovery rate from human fibroblasts. By combining a 3 <i>m</i>/<i>z</i> isolation window with short transients to improve specificity and signal-to-noise for proteoforms >30 kDa, we demonstrate improving proteome coverage by capturing 439 proteoforms in the 30–60 kDa range. Three different data acquisition strategies were compared and resulted in the identification of many proteoforms not observed in replicate data-dependent experiments. Notably, the data set is reported with updated metrics and tools including a new viewer and assignment of permanent proteoform record identifiers for inclusion of highly characterized proteoforms (i.e., those with C-scores >40) in a repository curated by the Consortium for Top-Down Proteomics

Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer

Over the past decade, developments in high resolution mass spectrometry have enabled the high throughput analysis of intact proteins from complex proteomes, leading to the identification of thousands of proteoforms. Several previous reports on top-down proteomics (TDP) relied on hybrid ion trap–Fourier transform mass spectrometers combined with data-dependent acquisition strategies. To further reduce TDP to practice, we use a quadrupole-Orbitrap instrument coupled with software for proteoform-dependent data acquisition to identify and characterize nearly 2000 proteoforms at a 1% false discovery rate from human fibroblasts. By combining a 3 <i>m</i>/<i>z</i> isolation window with short transients to improve specificity and signal-to-noise for proteoforms >30 kDa, we demonstrate improving proteome coverage by capturing 439 proteoforms in the 30–60 kDa range. Three different data acquisition strategies were compared and resulted in the identification of many proteoforms not observed in replicate data-dependent experiments. Notably, the data set is reported with updated metrics and tools including a new viewer and assignment of permanent proteoform record identifiers for inclusion of highly characterized proteoforms (i.e., those with C-scores >40) in a repository curated by the Consortium for Top-Down Proteomics

Native Electron Capture Dissociation Maps to Iron-Binding Channels in Horse Spleen Ferritin

Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome <i>c</i> dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome <i>c</i> dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-<i>m</i>/<i>z</i> quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced <i>c</i>-type fragment ions. Similar to the previously reported results on cytochrome <i>c</i>, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin

Native GELFrEE: A New Separation Technique for Biomolecular Assemblies

The cadre of protein complexes in cells performs an array of functions necessary for life. Their varied structures are foundational to their ability to perform biological functions, lending great import to the elucidation of complex composition and dynamics. Native separation techniques that are operative on low sample amounts and provide high resolution are necessary to gain valuable data on endogenous complexes. Here, we detail and optimize the use of tube gel separations to produce samples proven compatible with native, multistage mass spectrometry (nMS/MS). We find that a continuous system (i.e., no stacking gel) with a gradient in its extent of cross-linking and use of the clear native buffer system performs well for both fractionation and native mass spectrometry of heart extracts and a fungal secretome. This integrated advance in separations and nMS/MS offers the prospect of untargeted proteomics at the next hierarchical level of protein organization in biology

Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion–photon, ion–ion, and ion–neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS² spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies