Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes.

COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.

TLR2 senses the SARS-CoV-2 envelope protein to produce inflammatory cytokines.

The innate immune response is critical for recognizing and controlling infections through the release of cytokines and chemokines. However, severe pathology during some infections, including SARS-CoV-2, is driven by hyperactive cytokine release, or a cytokine storm. The innate sensors that activate production of proinflammatory cytokines and chemokines during COVID-19 remain poorly characterized. In the present study, we show that both TLR2 and MYD88 expression were associated with COVID-19 disease severity. Mechanistically, TLR2 and Myd88 were required for ß-coronavirus-induced inflammatory responses, and TLR2-dependent signaling induced the production of proinflammatory cytokines during coronavirus infection independent of viral entry. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand. In addition, blocking TLR2 signaling in vivo provided protection against the pathogenesis of SARS-CoV-2 infection. Overall, our study provides a critical understanding of the molecular mechanism of ß-coronavirus sensing and inflammatory cytokine production, which opens new avenues for therapeutic strategies to counteract the ongoing COVID-19 pandemic.

Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity.

Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPR(mt)), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPR(mt) signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPR(mt) induction, while gain of function is sufficient to extend lifespan in a UPR(mt)-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPR(mt) signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.

The Convergence of Systems and Reductionist Approaches in Complex Trait Analysis.

Research into the genetic and environmental factors behind complex trait variation has traditionally been segregated into distinct scientific camps. The reductionist approach aims to decrypt phenotypic variability bit by bit, founded on the underlying hypothesis that genome-to-phenome relations are largely constructed from the additive effects of their molecular players. In contrast, the systems approach aims to examine large-scale interactions of many components simultaneously, on the premise that interactions in gene networks can be both linear and non-linear. Both approaches are complementary, and they are becoming increasingly intertwined due to developments in gene editing tools, omics technologies, and population resources. Together, these strategies are beginning to drive the next era in complex trait research, paving the way to improve agriculture and toward more personalized medicine.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population.

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

Systems genetics of metabolism: the use of the BXD murine reference panel for multiscalar integration of traits.

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

NCoR1 is a conserved physiological modulator of muscle mass and oxidative function.

Transcriptional coregulators control the activity of many transcription factors and are thought to have wide-ranging effects on gene expression patterns. We show here that muscle-specific loss of nuclear receptor corepressor 1 (NCoR1) in mice leads to enhanced exercise endurance due to an increase of both muscle mass and of mitochondrial number and activity. The activation of selected transcription factors that control muscle function, such as MEF2, PPARß/δ, and ERRs, underpins these phenotypic alterations. NCoR1 levels are decreased in conditions that require fat oxidation, resetting transcriptional programs to boost oxidative metabolism. Knockdown of gei-8, the sole C. elegans NCoR homolog, also robustly increased muscle mitochondria and respiration, suggesting conservation of NCoR1 function. Collectively, our data suggest that NCoR1 plays an adaptive role in muscle physiology and that interference with NCoR1 action could be used to improve muscle function.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

Deep spatial profiling of Venezuelan equine encephalitis virus reveals increased genetic diversity amidst neuroinflammation and cell death during brain infection.

Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance.

PCfun: a hybrid computational framework for systematic characterization of protein complex function.

In molecular biology, it is a general assumption that the ensemble of expressed molecules, their activities and interactions determine biological function, cellular states and phenotypes. Stable protein complexes-or macromolecular machines-are, in turn, the key functional entities mediating and modulating most biological processes. Although identifying protein complexes and their subunit composition can now be done inexpensively and at scale, determining their function remains challenging and labor intensive. This study describes Protein Complex Function predictor (PCfun), the first computational framework for the systematic annotation of protein complex functions using Gene Ontology (GO) terms. PCfun is built upon a word embedding using natural language processing techniques based on 1 million open access PubMed Central articles. Specifically, PCfun leverages two approaches for accurately identifying protein complex function, including: (i) an unsupervised approach that obtains the nearest neighbor (NN) GO term word vectors for a protein complex query vector and (ii) a supervised approach using Random Forest (RF) models trained specifically for recovering the GO terms of protein complex queries described in the CORUM protein complex database. PCfun consolidates both approaches by performing a hypergeometric statistical test to enrich the top NN GO terms within the child terms of the GO terms predicted by the RF models. The documentation and implementation of the PCfun package are available at https://github.com/sharmavaruns/PCfun. We anticipate that PCfun will serve as a useful tool and novel paradigm for the large-scale characterization of protein complex function.

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II.

Variable temperature electrospray mass spectrometry is useful for multiplexed measurements of the thermal stabilities of biomolecules, but the ionization process can be disrupted by aggregation-prone proteins/complexes that have irreversible unfolding transitions. Resistively heating solutions containing a mixture of bovine carbonic anhydrase II (BCAII), a CO2 fixing enzyme involved in many biochemical pathways, and cytochrome c leads to complete loss of carbonic anhydrase signal and a significant reduction in cytochrome c signal above ∼72 °C due to aggregation. In contrast, when the tips of borosilicate glass nanoelectrospray emitters are heated with a laser, complete thermal denaturation curves for both proteins are obtained in <1 minute. The simultaneous measurements of the melting temperature of BCAII and BCAII bound to bicarbonate reveal that the bicarbonate stabilizes the folded form of this protein by ∼6.4 °C. Moreover, the temperature dependences of different bicarbonate loss pathways are obtained. Although protein analytes are directly heated by the laser for only 140 ms, heat conduction further up the emitter leads to a total analyte heating time of ∼41 s. Pulsed laser heating experiments could reduce this time to ∼0.5 s for protein aggregation that occurs on a faster time scale. Laser heating provides a powerful method for studying the detailed mechanisms of cofactor/ligand loss with increasing temperature and promises a new tool for studying the effect of ligands, drugs, growth conditions, buffer additives, or other treatments on the stabilities of aggregation-prone biomolecules.

Ion emission from 1-10 MDa salt clusters: individual charge state resolution with charge detection mass spectrometry.

Salt cluster ions produced by electrospray ionization are used for mass calibration and fundamental investigations into cluster stability and charge separation processes. However, previous studies have been limited to relatively small clusters owing to the heterogeneity associated with large, multiply-charged clusters that leads to unresolved signals in conventional m/z spectra. Here, charge detection mass spectrometry is used to measure both the mass and charge distributions of positively charged clusters of KCl, CaCl2, and LaCl3 with masses between ∼1 and 10 MDa by dynamically measuring the energy per charge, m/z, charge, and mass of simultaneously trapped individual ions throughout a 1 s trapping time. The extent of remaining hydration on the clusters, determined from the change in the frequency of ion motion with time as a result of residual water loss, follows the order KCl < CaCl2 < LaCl3, and is significantly lower than that of a pure water nanodrop, consistent with tighter water binding to the more highly charged cations in these clusters. The number of ion emission events from these clusters also follows this same trend, indicating that water at the cluster surface facilitates charge loss. A new frequency-based method to determine the magnitude of the charge loss resulting from individual ion emission events clearly resolves losses of +1 and +2 ions. Achieving this individual charge state resolution for ion emission events is an important advance in obtaining information about the late stages of bare gaseous ions formation. Future experiments on more hydrated clusters are expected to lead to a better understanding of ion formation in electrospray ionization.

Are Hydroxyl Radicals Spontaneously Generated in Unactivated Water Droplets?

Spontaneous ionization/breakup of water at the surface of aqueous droplets has been reported with evidence ranging from formation of hydrogen peroxide and hydroxyl radicals, indicated by ions at m/z 36 attributed to OH•-H3O+ or (H2O-OH2)+• as well as oxidation products of radical scavengers in mass spectra of water droplets formed by pneumatic nebulization. Here, aqueous droplets are formed both by nanoelectrospray, which produces highly charged nanodrops with initial diameters ~100 nm, and a vibrating mesh nebulizer, which produces 2 - 20 µm droplets that are less highly charged. The lifetimes of these droplets range from 10s of µs to 560 ms and the surface-to-volume ratios span ~100-fold range. No ions at m/z 36 are detected with pure water, nor are significant oxidation products for the two radical scavengers that were previously reported to be formed in high abundance. These and other results indicate that prior conclusions about spontaneous hydroxyl radical formation in unactivated water droplets are not supported by the evidence and that water appears to be stable at droplet surfaces over a wide range of droplet size, charge and lifetime.

Variable Mixing with Theta Emitter Mass Spectrometry: Changing Solution Flow Rates with Emitter Position.

Two solutions can be rapidly mixed using theta glass emitters, with products measured using electrospray ionization mass spectrometry. The relative flow rates of the two emitter channels can be measured using different calibration compounds in each channel, or the flow rates are often assumed to be the same. The relative flow rates of each channel can be essentially the same when the emitters are positioned directly in front of the capillary entrance of a mass spectrometer, but the relative flow rates can be varied by up to 3 orders of magnitude by moving the position of the emitter tip ±1 cm in a direction that is perpendicular to the inner divider. Results of the emitter position on the different concentrations of reagents in the initially formed electrospray droplets are demonstrated through protein denaturation using a supercharging reagent as well as two different bimolecular reactions. The average charge state of myoglobin changed from +7.8 to +13.8 when 2.5% sulfolane was mixed with a 200 mM ammonium acetate solution containing the protein when the position of the emitter was scanned in front of the mass spectrometer inlet. The conversion ratio of a bimolecular reaction was changed from 0.98 to 0.04 with varying emitter positions. These results show that the relative flow rates must be carefully monitored because the droplet composition depends strongly on the position of the theta glass emitters. This method can be used to measure the dependence of reaction kinetics on different solution concentrations by using a single emitter and only two solutions.

Combined Multiharmonic Frequency Analysis for Improved Dynamic Energy Measurements and Accuracy in Charge Detection Mass Spectrometry.

The ability to determine ion energies in electrostatic ion-trap-based charge detection mass spectrometry (CDMS) experiments is important for the accurate measurement of individual ion m/z, charge, and mass. Dynamic energy measurements throughout the time an ion is trapped take advantage of the relationship between ion energy and the harmonic amplitude ratio (HAR) composed from the fundamental and second harmonic amplitudes in the Fourier transform of the ion signal. This method eliminates the need for energy-filtering optics in CDMS and makes it possible to measure energy lost in collisions and changes in ion masses due to dissociation. However, the accuracy of the energy measurement depends on the signal-to-noise ratio (S/N) of the amplitudes used to determine the HAR. Here, a major improvement to this HAR-based dynamic energy measurement method is achieved using HARs composed of higher-order harmonics in addition to the fundamental and second harmonic to determine ion energies. This combined harmonic amplitude ratios for precision energy refinement (CHARPER) method is applied to the analysis of a 103 nm polystyrene nanoparticle ion (359.7 MDa, m/z = 308,300) and the energy resolution (3140) and effective mass resolution (730) achieved are the best yet demonstrated in electrostatic ion-trap-based CDMS. The CHARPER method applied to an ensemble of several thousand adeno-associated virus ion signals also results in higher mass resolution compared to the basic HAR method, making it possible to resolve additional features in the composite mass histogram.

Dynamic Energy Measurements in Charge Detection Mass Spectrometry Eliminate Adverse Effects of Ion-Ion Interactions.

Ion-ion interactions in charge detection mass spectrometers that use electrostatic traps to measure masses of individual ions have not been reported previously, although ion trajectory simulations have shown that these types of interactions affect ion energies and thereby degrade measurement performance. Here, examples of interactions between simultaneously trapped ions that have masses ranging from ca. 2 to 350 MDa and ca. 100 to 1000 charges are studied in detail using a dynamic measurement method that makes it possible to track the evolution of the mass, charge, and energy of individual ions over their trapping lifetimes. Signals from ions that have similar oscillation frequencies can have overlapping spectral leakage artifacts that result in slightly increased uncertainties in the mass determination, but these effects can be mitigated by the careful choice of parameters used in the short-time Fourier transform analysis. Energy transfers between physically interacting ions are also observed and quantified with individual ion energy measurement resolution as high as ∼950. The mass and charge of interacting ions do not change, and their corresponding measurement uncertainties are equivalent to ions that do not undergo physical interactions. Simultaneous trapping of multiple ions in CDMS can greatly decrease the acquisition time necessary to accumulate a statistically meaningful number of individual ion measurements. These results demonstrate that while ion-ion interactions can occur when multiple ions are trapped, they have negligible effects on mass accuracy when using the dynamic measurement method.

Endotyping pediatric obesity-related asthma: Contribution of anthropometrics, metabolism, nutrients, and CD4+ lymphocytes to pulmonary function.

BACKGROUND: Obesity-related complications including visceral fat, metabolic abnormalities, nutrient deficiencies, and immune perturbations are interdependent but have been individually associated with childhood asthma. OBJECTIVE: We sought to endotype childhood obesity-related asthma by quantifying contributions of obesity-related complications to symptoms and pulmonary function. METHODS: Multiomics analysis using Similarity Network Fusion followed by mediation analysis were performed to quantify prediction of obese asthma phenotype by different combinations of anthropometric, metabolic, nutrient, and TH-cell transcriptome and DNA methylome data sets. RESULTS: Two clusters (n = 28 and 26) distinct in their anthropometric (neck and midarm circumference, waist to hip ratio [WHR], and body mass index [BMI] z score), metabolic, nutrient, and TH-cell transcriptome and DNA methylome footprint predicted 5 or more pulmonary function indices across 7 different data set combinations. Metabolic measures attenuated the association of neck, WHR, and BMI z score with FEV1/forced vital capacity (FVC) ratio and expiratory reserve volume (ERV), of neck, midarm, and BMI z score with functional residual capacity, but only of WHR with inspiratory capacity. Nutrient levels attenuated the association of neck, midarm circumference, and BMI z score with functional residual capacity, and of WHR with FEV1/FVC ratio, ERV, and inspiratory capacity. TH-cell transcriptome attenuated the association of all 4 anthropometric measures with FEV1/FVC ratio, but only of WHR with ERV and inspiratory capacity. The DNA methylome attenuated the association of all 4 anthropometric measures with FEV1/FVC ratio and ERV, but only of WHR with inspiratory capacity. CONCLUSIONS: Anthropometric, metabolic, nutrient, and immune perturbations have individual but interdependent contributions to obese asthma phenotype, with the most consistent effect of WHR, highlighting the role of truncal adiposity in endotyping childhood obesity-related asthma.

Clinical outcomes of the use of unidirectional barbed sutures in gastrointestinal surgery for dogs and cats: A retrospective study.

OBJECTIVE: To report the clinical outcomes of gastrointestinal surgery using unidirectional barbed sutures in single-layer appositional closure in dogs and cats. STUDY DESIGN: Retrospective and descriptive study. SAMPLE POPULATION: Twenty-six client-owned dogs; three client-owned cats. METHODS: Medical records of dogs and cats that received gastrointestinal surgery closed with unidirectional barbed sutures were reviewed to collect information on signalment, physical examinations, diagnostics, surgical procedures, and complications. Short- and long-term follow-up information was collected from the medical records, the owners, or the referring veterinarians. RESULTS: Six gastrotomies, 21 enterotomies, and nine enterectomies were closed with a simple continuous pattern with unidirectional barbed glycomer 631 sutures. Nine dogs had multiple surgical sites closed with unidirectional barbed sutures. None of the cases in the study developed leakage, dehiscence, or septic peritonitis during the 14-day short-term follow up. Long-term follow up information was collected for 19 patients. The median long-term follow-up time was 1076 days (range: 20-2179 days). Two dogs had intestinal obstruction due to strictures at the surgical site 20 and 27 days after surgery. Both were resolved with an enterectomy of the original surgical site. CONCLUSION: Unidirectional barbed suture was not associated with a risk of leakage or dehiscence after gastrointestinal surgery in dogs and cats. However, strictures may develop in the long term. CLINICAL SIGNIFICANCE: Unidirectional barbed sutures can be used during gastrointestinal surgery in client-owned dogs and cats. Further investigation of the role of unidirectional barbed sutures leading to abscess, fibrosis, or stricture is necessary.

Characterizing Heterogeneous Mixtures of Assembled States of the Tobacco Mosaic Virus Using Charge Detection Mass Spectrometry.

The tobacco mosaic viral capsid protein (TMV) is a frequent target for derivatization for myriad applications, including drug delivery, biosensing, and light harvesting. However, solutions of the stacked disk assembly state of TMV are difficult to characterize quantitatively due to their large size and multiple assembled states. Charge detection mass spectrometry (CDMS) addresses the need to characterize heterogeneous populations of large protein complexes in solution quickly and accurately. Using CDMS, previously unobserved assembly states of TMV, including 16-monomer disks and odd-numbered disk stacks, have been characterized. We additionally employed a peptide-protein conjugation reaction in conjunction with CDMS to demonstrate that modified TMV proteins do not redistribute between disks. Finally, this technique was used to discriminate between protein complexes of near-identical mass but different configurations. We have gained a greater understanding of the behavior of TMV, a protein used across a broad variety of fields and applications, in the solution state.

Laser Heating Nanoelectrospray Emitters for Fast Protein Melting Measurements with Mass Spectrometry.

Temperature-controlled nanoelectrospray ionization has been used to measure heat-induced conformational changes of biomolecules by mass spectrometry, but long thermal equilibration times associated with heating or cooling an entire emitter limit how fast these data can be acquired. Here, the tip of a borosilicate electrospray emitter is heated using 10.6 µm light from an unfocused CO2 laser. At 1.2 W, the solution inside the emitter tip can be heated from room temperature to a steady-state temperature of 78.2 ± 2.5 °C in less than 0.5 s and cools from 82.6 ± 0.6 °C back to room temperature within 4 s. The time required to establish a steady-state temperature is more than 100-fold faster than that required for a resistively heated emitter due to the low thermal mass. Protein unfolding curves measured as a function of laser power can be acquired in ∼40 s compared to a resistively heated apparatus that required ∼21 min to acquire similar data. Laser power is calibrated to temperature by comparisons of the average charge state of the protein cytochrome c measured with laser heating and with resistive heating. This laser heating method is applied to a three-component protein mixture to demonstrate the ability to rapidly acquire melting temperatures of proteins in mixtures. The ability to rapidly assess the thermal stabilities of multiple proteins simultaneously shows significant promise for coupling temperature-controlled electrospray ionization (ESI) to separation techniques, providing a high-throughput method for determining the effects of solution composition, drug binding, or sequence mutations on protein thermal stability.