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Blood-based biomarkers for Alzheimer's disease (AD) are urgently needed. Here, four plasma biomarkers were measured at baseline in a community-based cohort followed over 17 years, and the association with clinical AD risk was determined.Amyloid beta (Aβ) misfolding status as a structure-based biomarker as well as phosphorylated tau 181 (P-tau181), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) concentration levels were determined at baseline in heparin plasma from 68 participants who were diagnosed with AD and 240 controls without dementia diagnosis throughout follow-up.Aβ misfolding exhibited high disease prediction accuracy of AD diagnosis within 17 years. Among the concentration markers, GFAP showed the best performance, followed by NfL and P-tau181. The combination of Aβ misfolding and GFAP increased the accuracy.Aβ misfolding and GFAP showed a strong ability to predict clinical AD risk and may be important early AD risk markers. Aβ misfolding illustrated its potential as a prescreening tool for AD risk stratification in older adults.

Blood biomarkers for Alzheimer's disease (AD) are the future of AD risk assessment. The aim of this study was to determine the association between plasma-measured phosphorylated tau (p-tau181), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) levels and risk of clinical AD incidence with consideration to the impact of cardiovascular health.Within a community-based cohort, biomarker levels were measured at baseline using single molecule array technology in 768 participants (aged 50-75) followed over 17 years. Associations among biomarkers and AD, vascular dementia, and mixed dementia incidence were assessed.GFAP was associated with clinical AD incidence even more than a decade before diagnosis (9-17 years), while p-tau181 and NfL were associated with more intermediate AD risk (within 9 years). Significant interaction was detected between cardiovascular health and p-tau181/NfL.GFAP may be an early AD biomarker increasing before p-tau181 and NfL and the effect modifying role of cardiovascular health should be considered in biomarker risk stratification.

This study assessed whether in a population with comorbidity of neurodegenerative and cerebrovascular disease (mixed pathology) the association of glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), and phosphorylated tau181 (p-tau181) with dementia risk varied depending on levels of total cholesterol and apolipoprotein E (APOE) ε4 genotype.Plasma biomarkers were measured using Simoa technology in 768 participants of a nested case-control study embedded within an ongoing population-based cohort. Logistic and spline regression models, and receiver operating characteristic curves were calculated.The strength of the association between GFAP and NfL with risk of a clinical diagnosis of dementia changed depending on cholesterol levels and on APOE ε4 genotype. No significant association was seen with p-tau181.In individuals with mixed pathology blood GFAP and NfL are better predictors of dementia risk than p-tau181, and their associations with dementia risk are amplified by hypercholesterolemia, also depending on APOE ε4 genotype.Cholesterol levels changed the association of blood biomarkers with dementia risk. Blood biomarkers seem to perform differently in community- and clinic-based cohorts. Neurofilament light chain might be a biomarker candidate for dementia risk after stroke.

Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)—including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn—accumulate in Lewy bodies (LBs) in different regions of the Parkinson’s disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-021-02329-9.

Therapeutic decisions in lung cancer critically depend on the determination of histologic types and oncogene mutations. Therefore, tumor samples are subjected to standard histologic and immunohistochemical analyses and examined for relevant mutations using comprehensive molecular diagnostics. In this study, an alternative diagnostic approach for automatic and label-free detection of mutations in lung adenocarcinoma tissue using quantum cascade laser-based infrared imaging is presented. For this purpose, a five-step supervised classification algorithm was developed, which was not only able to detect tissue types and tumor lesions, but also the tumor type and mutation status of adenocarcinomas. Tumor detection was verified on a data set of 214 patient samples with a specificity of 97% and a sensitivity of 95%. Furthermore, histology typing was verified on samples from 203 of the 214 patients with a specificity of 97% and a sensitivity of 94% for adenocarcinoma. The most frequently occurring mutations in adenocarcinoma (KRAS, EGFR, and TP53) were differentiated by this technique. Detection of mutations was verified in 60 patient samples from the data set with a sensitivity and specificity of 95% for each mutation. This demonstrates that quantum cascade laser infrared imaging can be used to analyze morphologic differences as well as molecular changes. Therefore, this single, one-step measurement provides comprehensive diagnostics of lung cancer histology types and most frequent mutations.

Background: Digital pathology, in its primary meaning, describes the utilization of computer screens to view scanned histology slides. Digitized tissue sections can be easily shared for a second opinion. In addition, it allows tissue image analysis using specialized software to identify and measure events previously observed by a human observer. These tissue-based readouts were highly reproducible and precise. Digital pathology has developed over the years through new technologies. Currently, the most discussed development is the application of artificial intelligence to automatically analyze tissue images. However, even new label-free imaging technologies are being developed to allow imaging of tissues by means of their molecular composition. Summary: This review provides a summary of the current state-of-the-art and future digital pathologies. Developments in the last few years have been presented and discussed. In particular, the review provides an outlook on interesting new technologies (e.g., infrared imaging), which would allow for deeper understanding and analysis of tissue thin sections beyond conventional histopathology. Key Messages: In digital pathology, mathematical methods are used to analyze images and draw conclusions about diseases and their progression. New innovative methods and techniques (e.g., label-free infrared imaging) will bring significant changes in the field in the coming years.

Background Follow-up recommendations for patients with nonmuscle invasive bladder cancer (NMIBC) are largely based upon expert opinion. A growing body of evidence suggests that current follow-up strategies for bladder cancer patients with low and intermediate risk represent overdiagnosis and may lead to overtreatment. The goal of this study is to explore the options of a noninvasive follow-up in patients with pTa G1-2/low-grade NMIBC. Methods The risks and options for a urine marker-guided, noninvasive follow-up of patients with pTa G1-2/low-grade NMIBC were defined and the study design for a prospective randomized trial (UroFollow) was developed based upon the current literature. Results The investigators postulated that follow-up of patients with pTa G1-2/low-grade NMIBC requires a high sensitivity of urinary tumor markers. However, data from prospective studies with prediagnostic urine samples are scarce, even for approved markers, and cross-sectional studies with symptomatic patients overestimate the sensitivity. So far, cell-based markers (e.g., uCyt+ and UroVysion) in urine appeared to have higher sensitivities and specificities in low-grade NMIBC than urine cytology and markers analyzing soluble tumor-associated antigens. Marker panels are more sensitive than single-marker approaches at the expense of a lower specificity. Given a prospective randomized comparison with a marker sensitivity of 80% compared to usual care with cystoscopy, the sample size calculation yielded that 62 to 185 patients under study per arm are needed depending on different recurrence rates. Conclusions Based upon these findings the UroFollow trial has been designed as a prospective randomized study comparing a noninvasive marker-based (UroVysion, NMP22, urine cytology, and ultrasound) follow-up with the current standard of care over a period of 3 years.

Background We evaluated Aβ misfolding in combination with Aβ42/40 ratio as a prognostic tool for future clinical progression to mild cognitive impairment (MCI) or dementia due to Alzheimer’s disease (AD) in individuals with subjective cognitive decline (SCD). Methods Baseline plasma samples (n = 203) from SCD subjects in the SCIENCe project and Amsterdam Dementia Cohort (age 61 ± 9 years; 57% male, mean follow-up time 2.7 years) were analyzed using immuno-infrared-sensor technology. Within 6 years of follow-up, 22 (11%) individuals progressed to MCI or dementia due to AD. Sensor readout values > 1646 cm− 1 reflected normal Aβ folding; readouts at ≤ 1646 cm− 1 reflected low and at − 1 high misfolding. We used Cox proportional hazard models to quantify Aβ misfolding as a prognostic biomarker for progression to MCI and dementia due to AD. The accuracy of the predicted development of MCI/AD was determined by time-dependent receiver operating characteristic (t-ROC) curve analyses that take individual follow-up and conversion times into account. Statistical models were adjusted for age, sex, and APOEε4 status. Additionally, plasma Aβ42/40 data measured by SIMOA were statistically analyzed and compared. Results All 22 patients who converted to MCI or AD-dementia within 6 years exhibited Aβ misfolding at baseline. Cox analyses revealed a hazard ratio (HR) of 19 (95% confidence interval [CI] 2.2–157.8) for future conversion of SCD subjects with high misfolding and of 11 (95% CI 1.0–110.1) for those with low misfolding. T-ROC curve analyses yielded an area under the curve (AUC) of 0.94 (95% CI 0.86–1.00; 6-year follow-up) for Aβ misfolding in an age, sex, and APOEε4 model. A similar model with plasma Aβ42/40 ratio yielded an AUC of 0.92 (95% CI, 0.82–1.00). The AUC increased to 0.99 (95% CI, 0.99–1.00) after inclusion of both Aβ misfolding and the Aβ42/40 ratio. Conclusions A panel of structure- and concentration-based plasma amyloid biomarkers may predict conversion to clinical MCI and dementia due to AD in cognitively unimpaired subjects. These plasma biomarkers provide a noninvasive and cost-effective alternative for screening early AD pathological changes. Follow-up studies and external validation in larger cohorts are in progress for further validation of our findings.

Time-resolved step-scan FTIR difference-specroscopy allows monitoring of protein-reactions with nanoseconds time-resolution at atomic resolution. The technique monitors reaction of prosthetic groups, like cis-trans isomerization, movements of the protein backbone and protonation changes or H-bond changes of aminoacids.

In recent years, deep learning has been the key driver of breakthrough developments in computational pathology and other image based approaches that support medical diagnosis and treatment. The underlying neural networks as inherent black boxes lack transparency, and are often accompanied by approaches to explain their output. However, formally defining explainability has been a notorious unsolved riddle. Here, we introduce a hypothesis-based framework for falsifiable explanations of machine learning models. A falsifiable explanation is a hypothesis that connects an intermediate space induced by the model with the sample from which the data originate. We instantiate this framework in a computational pathology setting using label-free infrared microscopy. The intermediate space is an activation map, which is trained with an inductive bias to localize tumor. An explanation is constituted by hypothesizing that activation corresponds to tumor and associated structures, which we validate by histological staining as an independent secondary experiment.

Background To understand the potential for early intervention and prevention measures in Alzheimer’s disease, the association between risk factors and early pathological change needs to be assessed. Hence, the aim of this study was to determine whether risk factors of Alzheimer’s clinical syndrome (clinical AD), such as body mass index (BMI), are associated with Aβ misfolding in blood, a strong risk marker for AD among older adults. Methods Information on risk factors and blood samples were collected at baseline in the ESTHER study, a population-based cohort study of older adults (age 50–75 years) in Germany. Aβ misfolding in blood plasma was analyzed using an immuno-infrared-sensor in a total of 872 participants in a nested case-control design among incident dementia cases and matched controls. Associations between risk factors and Aβ misfolding were assessed by multiple logistic regression. For comparison, the association between the risk factors and AD incidence during 17 years of follow-up was investigated in parallel among 5987 cohort participants. Results An inverse association with Aβ misfolding was seen for BMI at age 50 based on reported weight history (aOR 0.64, 95% CI 0.43–0.96, p = 0.03). Similar but not statistically significant associations were seen for BMI at baseline (i.e., mean age 68) and at age 40. No statistically significant associations with Aβ misfolding were found for other risk factors, such as diabetes, smoking, and physical activity. On the other hand, low physical activity was associated with a significantly reduced risk of developing clinical AD compared to physical inactivity. Conclusions Our results support that AD pathology may be detectable and associated with reduced weight even in middle adulthood, many years before clinical diagnosis of AD. Physical activity might reduce the risk of onset of AD symptoms.

Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein–protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target., By combining the incorporation of unnatural amino acids, click chemistry, FRET and EPR distance measurements, protein modeling and biomolecular simulations, we obtained an unambiguous Ras dimer structural model and disrupt the dimer by mutagenesis.

A label-free approach based on a highly reproducible and stable workflow allows for quantitative proteome analysis . Due to advantages compared to labeling methods, the label-free approach has the potential to measure unlimited samples from clinical specimen monitoring and comparing thousands of proteins. The presented label-free workflow includes a new sample preparation technique depending on automatic annotation and tissue isolation via FTIR-guided laser microdissection, in-solution digestion, LC-MS/MS analyses, data evaluation by means of Proteome Discoverer and Progenesis software, and verification of differential proteins. We successfully applied this workflow in a proteomics study analyzing human cystitis and high-grade urothelial carcinoma tissue regarding the identification of a diagnostic tissue biomarker. The differential analysis of only 1 mm

Alzheimer’s disease cannot be cured today. In addition to a drug, a predictive blood biomarker is urgently needed to identify high-risk subjects in early cognitively less impaired stages. Here, a biomarker is presented that uses the secondary structure distribution of Aβ-peptides in blood measured by the immuno-infrared sensor. Using this structure-based biomarker clinical conversion of symptomless subjects at baseline were predicted in a follow-up study 14 years before manifestation.

Optogenetics uses light‐sensitive proteins, so‐called optogenetic tools, for highly precise spatiotemporal control of cellular states and signals. The major limitations of such tools include the overlap of excitation spectra, phototoxicity, and lack of sensitivity. The protein characterized in this study, the Japanese lamprey parapinopsin, which we named UVLamP, is a promising optogenetic tool to overcome these limitations. Using a hybrid strategy combining molecular, cellular, electrophysiological, and computational methods we elucidated a structural model of the dark state and probed the optogenetic potential of UVLamP. Interestingly, it is the first described bistable vertebrate opsin that has a charged amino acid interacting with the Schiff base in the dark state, that has no relevance for its photoreaction. UVLamP is a bistable UV‐sensitive opsin that allows for precise and sustained optogenetic control of G protein‐coupled receptor (GPCR) pathways and can be switched on, but more importantly also off within milliseconds via lowintensity short light pulses. UVLamP exhibits an extremely narrow excitation spectrum in the UV range allowing for sustained activation of the Gi/o pathway with a millisecond UV light pulse. Its sustained pathway activation can be switched off, surprisingly also with a millisecond blue light pulse, minimizing phototoxicity. Thus, UVLamP serves as a minimally invasive, narrow‐bandwidth probe for controlling the Gi/o pathway, allowing for combinatorial use with multiple optogenetic tools or sensors. Because UVLamP activated Gi/o signals are generally inhibitory and decrease cellular activity, it has tremendous potential for health‐related applications such as relieving pain, blocking seizures, and delaying neurodegeneration., The Japanese lamprey parapinopsin (“UVLamP”) serves as a minimally invasive, narrow‐bandwidth, bistable next‐generation optogenetic probe for controlling the Gi/o pathway. A millisecond UV light pulse allows sustained pathway activation that can be switched off with a millisecond blue light pulse on demand. The first structural model of parapinopsin in the dark state reveals novel interaction partners shedding light on mechanisms responsible for opsin bistability.

Significance Understanding the mechanisms of photoactivated biological processes facilitates the development of new molecular tools, engineered for specific optogenetic applications, allowing the control of neuronal activity with light. Here, we use a variety of experimental and theoretical techniques to examine the precise nature of the light-activated ion channel in one of the most important molecular species used in optogenetics, channelrhodopsin-2. Existing models for the photochemical and photophysical pathway after light absorption by the molecule fail to explain many aspects of its observed behavior, including the inactivation of the photocurrent under continuous illumination. We resolve this by proposing a branched photocycle explaining electrical and photochemical channel properties and establishing the structure of intermediates during channel turnover., Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

Introduction Alzheimer's disease (AD) diagnosis requires invasive CSF analysis or expensive brain imaging. Therefore, a minimal-invasive reliable and cost-effective blood test is requested to power large clinical AD trials at reduced screening failure. Methods We applied an immuno-infrared sensor to measure the amyloid-β (Aβ) and tau secondary structure distribution in plasma and CSF as structure-based biomarkers for AD (61 disease controls, 39 AD cases). Results Within a first diagnostic screening step, the structure-based Aβ blood biomarker supports AD identification with a sensitivity of 90%. In a second diagnostic validation step, the combined use of the structure-based CSF biomarkers Aβ and tau excluded false-positive cases which offers an overall specificity of 97%. Discussion The primary Aβ-based blood biomarker funnels individuals with suspected AD for subsequent validation of the diagnosis by structure-based combined analysis of the CSF biomarkers Aβ and tau. Our novel two-step recruitment strategy substantiates the diagnosis of AD with a likelihood of 29.

In recent years, gating and transient ion-pathway formation in the light-gated channelrhodopsins (ChRs) have been intensively studied. Despite these efforts, a profound understanding of the mechanistic details is still lacking. To track structural changes concomitant with the formation and subsequent collapse of the ion-conducting pore, we site-specifically introduced the artificial polarity-sensing probe p-azido-l-phenylalanine (azF) into several ChRs by amber stop codon suppression. The frequently used optogenetic actuator ReaChR (red-activatable ChR) exhibited the best expression properties of the wild type and the azF mutants. By exploiting the unique infrared spectral absorption of azF [ν

Raman spectroscopy can provide the biomolecular fingerprint of a cell in a label-free manner. Although a variety of clinical and biomedical applications have been demonstrated, the method remains largely a niche technology. The two main problems are the complexity of data acquisition and the complexity of data analysis. Generally, Raman measurements are performed manually and require a substantial amount of time. This, on the other hand, frequently results in a low number of samples and hence with questionable statistical evaluation. Here, we propose an automated high content screening Raman spectroscopy (HCS-RS) platform, which can perform a series of experiments without human interaction, significantly increasing the number of measured samples and making the measurement more reliable. The automated image processing of bright field images in combination with automatic spectral acquisition of the molecular fingerprint of cells exposed to different physiological conditions enables label-free high content screening applications. The performance of the developed HCS-RS platform is demonstrated by investigating the effect of panitumumab on SW48 and SW480 colorectal cancer cells with wild-type and mutated K-RAS, respectively, in a series of concentrations. Our result indicates that the increased content of panitumumab prohibits the activation of the MAP kinase of the colorectal cancer cells with wild-type K-RAS strongly, whereas there is no significant effect on the K-RAS mutated cells. Moreover, the relative amount of the panitumumab content present in the cells is determined from the Raman spectral information, which could be beneficial for personalized patient treatment.

3616 Background: MSI due to mismatch repair defects accounts for 15-20% of all CC, has high prognostic and predictive value and is broadly utilized in treatment decisions. Artificial intelligence (AI) integrated, label-free quantum cascade laser (QCL) based infrared (IR) imaging resolves spatial and molecular alterations such as MSI in unstained cancer tissue sections. We aimed to evaluate the method for microsatellite instability/stability (MSI/MSS) classification in samples from the prospective multicenter AIO CPP registry trial. Methods: Paraffin-embedded unstained cancer tissue slides from patients (pts.) participating in CPP were measured (avg. 30 min/slide) and analyzed. The cohort was split into training (train), test (test), and validation (vali) sets. Cancer regions were first preselected based on a self-developed convolutional neural network (CNN) CompSegNet (Schuhmacher, medrxiv 2021). A VGG-16 CNN then classified MSI/MSS in these regions. Endpoints were area under receiver operating characteristic (AUROC) and area under precision recall curve (AUPRC). Results: 547 pts. (train n=331, test n=69, vali n=147) were analyzed. The baseline characteristics for the sub-cohorts are illustrated in the table. Mutation (MT) status: RAS MT: train 30% / test 30% / vali 37%; BRAF MT: train 27% / test 23% / vali 14%. The preselection of cancer regions reached a validation AUROC of 1.0. The subsequent MSI/MSS classifier reached a validation AUROC of 0.9 and AUPRC of 0.74 (sensitivity 85%, specificity 84%). Conclusions: Our multicenter approach using AI integrated label-free IR imaging provides an automated, fast, and reliable classification for MSI/MSS with an AUROC of 0.9 (sensitivity 85%, specificity 84%) almost comparable to the present gold standard immunohistochemistry. The method described here requires less samples for training when compared to other AI approaches which could facilitate the development of prognostic/predictive classifiers in the setting of randomized controlled trials. This novel technique may support further understanding of the increasingly important MSI CC cohort and support treatment decisions e.g. in specific subgroups such as targetable fusions. We expect our approach to be a broadly applicable diagnostic tool in the future.[Table: see text]

Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.