Defining mitochondrial protein functions through deep multiomic profiling.

Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.

Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

Rapid Targeted Quantitation of Protein Overexpression with Direct Infusion Shotgun Proteome Analysis (DISPA-PRM).

While much effort has been placed on comprehensive quantitative proteome analysis, certain applications demand the measurement of only a few target proteins from complex systems. Traditional approaches to targeted proteomics rely on nanoliquid chromatography (nLC) and targeted mass spectrometry (MS) methods, e.g., parallel reaction monitoring (PRM). However, the time requirement for nLC can limit the throughput of targeted proteomics. To achieve rapid and high-throughput targeted methods, here we show that nLC separations can be eliminated and replaced with direct infusion shotgun proteome analysis (DISPA) using high-field asymmetric waveform ion mobility spectrometry (FAIMS) with PRM. We demonstrate the application of DISPA-PRM for rapid targeted quantification of bacterial enzymes utilized in the production of biofuels by monitoring temporal expression in 72 metabolically engineered bacterial cultures in less than 2.5 h, with a measured dynamic range >1200-fold. We conclude that DISPA-PRM presents a valuable innovative tool with results comparable to nLC-MS/MS, enabling fast and rapid detection of targeted proteins in complex mixtures.

ID-MAM: A Validated Identity and Multi-Attribute Monitoring Method for Commercial Release and Stability Testing of a Bispecific Antibody.

Post-translational modifications (PTMs) that impact the safety or efficacy of protein therapeutics are critical quality attributes (CQAs) that need to be controlled to ensure product quality. Peptide mapping with online mass spectrometry (MS) is a powerful tool that has been used for many years to monitor PTM CQAs during product development. However, operating peptide mapping methods with high-resolution mass spectrometers in GMP compliant, commercial quality control (QC) labs can be difficult. Peptide mapping is also required as an identity test in several countries. To address these two different needs, we utilized high-resolution peptide mapping for comprehensive characterization during development and then developed and validated a targeted multi-attribute monitoring (MAM) method using the low-resolution Waters QDa MS system with a fully automated data processing workflow that is suitable for identity (ID) testing, sequence variant control, and CQA quantitation in commercial QC labs. The ID-MAM method was validated for the quantitation of three selected PTM CQAs (CDR isomerization, Fc Met oxidation, and CDR Met oxidation) to ensure control of the oxidation and isomerization degradation pathways of a bispecific antibody (BsAb). This ID-MAM method was successfully validated in six labs (three analytical development and three QC labs) across four countries for commercial release and stability testing of a BsAb. CQA results obtained with the ID-MAM method were similar to results obtained using high-resolution peptide mapping, and the method was robust and reproducible. To our knowledge, this ID-MAM method is the first MS-based peptide mapping method implemented in GMP compliant QC labs for commercial release and stability testing of a biotherapeutic.

Multi-Omic Single-Shot Technology for Integrated Proteome and Lipidome Analysis.

Mass spectrometry (MS) serves as the centerpiece technology for proteome, lipidome, and metabolome analysis. To gain a better understanding of the multifaceted networks of myriad regulatory layers in complex organisms, integration of different multiomic layers is increasingly performed, including joint extraction methods of diverse biomolecular classes and comprehensive data analyses of different omics. Despite the versatility of MS systems, fractured methodology drives nearly all MS laboratories to specialize in analysis of a single ome at the exclusion of the others. Although liquid chromatography-mass spectrometry (LC-MS) analysis is similar for different biomolecular classes, the integration on the instrument level is lagging behind. The recent advancements in high flow proteomics enable us to take a first step towards integration of protein and lipid analysis. Here, we describe a technology to achieve broad and deep coverage of multiple molecular classes simultaneously through multi-omic single-shot technology (MOST), requiring only one column, one LC-MS instrument, and a simplified workflow. MOST achieved great robustness and reproducibility. Its application to a Saccharomyces cerevisiae study consisting of 20 conditions revealed 2842 protein groups and 325 lipids and potential molecular relationships.

Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome.

Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

Global Phosphoproteome Analysis Using High-Field Asymmetric Waveform Ion Mobility Spectrometry on a Hybrid Orbitrap Mass Spectrometer.

Mass spectrometry is the premier tool for identifying and quantifying protein phosphorylation on a global scale. Analysis of phosphopeptides requires enrichment, and even after the samples remain highly complex and exhibit broad dynamic range of abundance. Achieving maximal depth of coverage for phosphoproteomics therefore typically necessitates offline liquid chromatography prefractionation, a time-consuming and laborious approach. Here, we incorporate a recently commercialized aerodynamic high-field asymmetric waveform ion mobility spectrometry (FAIMS) device into the phosphoproteomic workflow. We characterize the effects of phosphorylation on the FAIMS separation, describe optimized compensation voltage settings for unlabeled phosphopeptides, and demonstrate the advantages of FAIMS-enabled gas-phase fractionation. Standard FAIMS single-shot analyses identified around 15-20% additional phosphorylation sites than control experiments without FAIMS. In comparison to liquid chromatography prefractionation, FAIMS experiments yielded similar or superior results when analyzing up to four discrete gas-phase fractions. Although using FAIMS led to a modest reduction in the precision of quantitative measurements when using label-free approaches, the data collected with FAIMS yielded a 26% increase in total reproducible measurements. Overall, we conclude that the new FAIMS technology is a valuable addition to any phosphoproteomic workflow, with greater benefits emerging from longer analyses and higher amounts of material.

Ribonucleic Acid Sequence Characterization by Negative Electron Transfer Dissociation Mass Spectrometry.

Modified oligonucleotides represent a promising avenue for drug development, with small interfering RNAs (siRNA) and microRNAs gaining traction in the therapeutic market. Mass spectrometry (MS)-based analysis offers many benefits for characterizing modified nucleic acids. Negative electron transfer dissociation (NETD) has proven valuable in sequencing oligonucleotide anions, particularly because it can retain modifications while generating sequence-informative fragments. We show that NETD can be successfully implemented on a widely available quadrupole-Orbitrap-linear ion trap mass spectrometer that uses a front-end glow discharge source to generate radical fluoranthene reagent cations. We characterize both unmodified and modified ribonucleic acids and present the first application of activated-ion negative electron transfer dissociation (AI-NETD) to nucleic acids. AI-NETD achieved 100% sequence coverage for both a 6-mer (5'-rGmUrArCmUrG-3') with 2'-O-methyl modifications and a 21-mer (5'-rCrArUrCrCrUrCrUrArGrArGrGrArUrArGrArArUrG-3'), the luciferase antisense siRNA. Both NETD and AI-NETD afforded complete sequence coverage of these molecules while maintaining a relatively low degree of undesired base-loss products and internal products relative to collision-based methods.

Proteomic Atlas of the Human Brain in Alzheimer's Disease.

The brain represents one of the most divergent and critical organs in the human body. Yet, it can be afflicted by a variety of neurodegenerative diseases specifically linked to aging, about which we lack a full biomolecular understanding of onset and progression, such as Alzheimer's disease (AD). Here we provide a proteomic resource comprising nine anatomically distinct sections from three aged individuals, across a spectrum of disease progression, categorized by quantity of neurofibrillary tangles. Using state-of-the-art mass spectrometry, we identify a core brain proteome that exhibits only small variance in expression, accompanied by a group of proteins that are highly differentially expressed in individual sections and broader regions. AD affected tissue exhibited slightly elevated levels of tau protein with similar relative expression to factors associated with the AD pathology. Substantial differences were identified between previous proteomic studies of mature adult brains and our aged cohort. Our findings suggest considerable value in examining specifically the brain proteome of aged human populations from a multiregional perspective. This resource can serve as a guide, as well as a point of reference for how specific regions of the brain are affected by aging and neurodegeneration.

Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample.

The dependence of capillary zone electrophoresis (CZE) separations on the charge state of the analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume was between 100 and 150 nL of a solution of phosphopeptides in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single-shot CZE-ESI-MS/MS-based phosphoproteome analysis. We found that the migration time for phosphopeptides is much longer than that for non-phosphopeptides and increased along with the number of phosphorylation sites on the peptides, as expected for the additional negative charges associated with the phosphate groups. We also investigated the phosphorylation site motifs; a number of motifs appeared in the CZE-ESI-MS/MS data but not in LC-ESI-MS/MS data, which suggested the complementary performance of the techniques. The data are available via ProteomeXchange with identifier PXD012888.

Islet proteomics reveals genetic variation in dopamine production resulting in altered insulin secretion.

The mouse is a critical model in diabetes research, but most research in mice has been limited to a small number of mouse strains and limited genetic variation. Using the eight founder strains and both sexes of the Collaborative Cross (C57BL/6J (B6), A/J, 129S1/SvImJ (129), NOD/ShiLtJ (NOD), NZO/HILtJ (NZO), PWK/PhJ (PWK), WSB/EiJ (WSB), and CAST/EiJ (CAST)), we investigated the genetic dependence of diabetes-related metabolic phenotypes and insulin secretion. We found that strain background is associated with an extraordinary range in body weight, plasma glucose, insulin, triglycerides, and insulin secretion. Our whole-islet proteomic analysis of the eight mouse strains demonstrates that genetic background exerts a strong influence on the islet proteome that can be linked to the differences in diabetes-related metabolic phenotypes and insulin secretion. We computed protein modules consisting of highly correlated proteins that enrich for biological pathways and provide a searchable database of the islet protein expression profiles. To validate the data resource, we identified tyrosine hydroxylase (Th), a key enzyme in catecholamine synthesis, as a protein that is highly expressed in ß-cells of PWK and CAST islets. We show that CAST islets synthesize elevated levels of dopamine, which suppresses insulin secretion. Prior studies, using only the B6 strain, concluded that adult mouse islets do not synthesize l-3,4-dihydroxyphenylalanine (l-DOPA), the product of Th and precursor of dopamine. Thus, the choice of the CAST strain, guided by our islet proteomic survey, was crucial for these discoveries. In summary, we provide a valuable data resource to the research community, and show that proteomic analysis identified a strain-specific pathway by which dopamine synthesized in ß-cells inhibits insulin secretion.

Maximizing Tandem Mass Spectrometry Acquisition Rates for Shotgun Proteomics.

Advances in tandem mass spectrometry (MS/MS) acquisition rate have steadily led to increased performance in shotgun proteomics experiments. To that end, contemporary mass spectrometers are outfitted with multiple analyzers allowing for the simultaneous collection of survey (MS1) and MS/MS spectra. In the latest generation Orbitrap hybrid, MS/MS scans can be acquired at a high rate using the dual cell linear ion trap analyzer, all while the next precursor is being dissociated in a collision cell and a MS1 scan is occurring in the Orbitrap. Often overlooked in these experiments is that the ion trap scan duration is highly variable and dependent upon precursor mass. Here, we examine the use of various static mass-to-charge ratio scan ranges for ion trap MS/MS acquisition and determine performance relative to conventional dynamic mass-to-charge ratio range scanning. We demonstrate that a fixed mass-to-charge ratio scan range can generate 12% more MS/MS scans and more unique peptide identifications as compared to the standard dynamic approach, respectively.

OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate.

Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.

Correction: Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

[This corrects the article DOI: 10.1371/journal.pgen.1006372.].

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3.

Purified human eosinophils treated for 18-24 h with IL-3 adopt a unique activated phenotype marked by increased reactivity to aggregated immunoglobulin-G (IgG). To characterize this phenotype, we quantified protein abundance and phosphorylation by multiplexed isobaric labeling combined with high-resolution mass spectrometry. Purified blood eosinophils of five individuals were treated with IL-3 or no cytokine for 20 h, and comparative data were obtained on abundance of 5385 proteins and phosphorylation at 7330 sites. The 1150 proteins that were significantly up-regulated ( q < 0.05, pairwise t test with Benjamini-Hochberg correction) by IL-3 included the IL3RA and CSF2RB subunits of the IL-3 receptor, the low-affinity receptor for IgG (FCGR2B), 96 proteins involved in protein translation, and 55 proteins involved in cytoskeleton organization. Among the 703 proteins that decreased were 78 mitochondrial proteins. Dynamic regulation of protein phosphorylation was detected at 4218 sites. These included multiple serines in CSF2RB; Y694 of STAT5, a key site of activating phosphorylation downstream of IL3RA/CSF2RB; and multiple sites in RPS6KA1, RPS6, and EIF4B, which are responsible for translational initiation. We conclude that IL-3 up-regulates overall protein synthesis and targets specific proteins for up-regulation, including its own receptor.

Ultra-High Pressure (>30,000 psi) Packing of Capillary Columns Enhancing Depth of Shotgun Proteomic Analyses.

Extreme sample complexity is an inherent challenge in shotgun proteomics that positions quality of chromatographic separations as one of the key determinants of attainable proteome coverage. In search of better separations, macroscopic physical characteristics of capillary columns, i.e., length and properties of stationary phase particles, are typically considered and optimized, while significance of packing bed morphology is frequently underappreciated. Here, we describe a technology that enables packing of capillary columns at excess of 30,000 psi and demonstrate that such columns exhibit reduced backpressure and remarkably reproducible chromatographic performance, improved on average by 23%. These enhancements afford up to 35% increase in the depth of commonplace bottom-up proteomic analyses, owning to augmented sensitivity and resolution of peptide separations and improvements in spectral quality. Our findings strongly corroborate advantages of ultra-high pressure packing of capillary columns for diverse shotgun proteomic workflows.

Production of Over 27 000 Peptide and Nearly 4400 Protein Identifications by Single-Shot Capillary-Zone Electrophoresis-Mass Spectrometry via Combination of a Very-Low-Electroosmosis Coated Capillary, a Third-Generation Electrokinetically-Pumped Sheath-Flow Nanospray Interface, an Orbitrap Fusion Lumos Tribrid Mass Spectrometer, and an Advanced-Peak-Determination Algorithm.

We show that capillary-zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) generates very large numbers of peptide and protein identifications (IDs) by combining four technologies: a separation capillary coated to generate very low electroosmosis, an electrokinetically pumped sheath-flow nanoelectrospray interface to produce high-sensitivity ionization, an Orbitrap Fusion Lumos Tribrid platform to provide high-speed analysis, and an advanced-peak-determination (APD) algorithm to take advantage of the mass spectrometer's data-acquisition speed. The use of the APD algorithm resulted in 2 times more identifications than the standard peak algorithm. We also investigated the effect of the isolation window, injection time, and loading amount. Optimization of these parameters produced over 27 000 peptide identifications and nearly 4400 protein-group identifications from 220 ng of K562-cell digest in a single 120 min run, which is 2.7 times more IDs produced by CZE-ESI-MS/MS than by the previous state-of-the-art technique.

Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer.

Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI-FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments of a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. A 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications.