Proteomic analysis on the scale that captures population and biological heterogeneity over hundreds to thousands of samples requires rapid mass spectrometry methods, which maximize instrument utilization (IU) and proteome coverage while maintaining precise and reproducible quantification. To achieve this, a short liquid chromatography gradient paired to rapid mass spectrometry data acquisition can be used to reproducibly quantify a moderate set of analytes. Highthroughput profiling at a limited depth is becoming an increasingly utilized strategy for tackling large sample sets but the time spent on loading the sample, flushing the column(s), and re-equilibrating the system reduces the ratio of meaningful data acquired to total operation time and IU. The dual-trap single-column configuration (DTSC) presented here maximizes IU in rapid analysis (15 min per sample) of blood and cell lysates by parallelizing trap column cleaning and sample loading and desalting with the analysis of the previous sample. We achieved 90% IU in low microflow (9.5 μL/min) analysis of blood while reproducibly quantifying 300−400 proteins and over 6000 precursor ions. The same IU was achieved for cell lysates and over 4000 proteins (3000 at CV below 20%) and 40,000 precursor ions were quantified at a rate of 15 min/sample. Thus, DTSC enables high-throughput epidemiological blood-based biomarker cohort studies and cell-based perturbation screening.

Background Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. Methods Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. Results Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide’s analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. Conclusion The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Objectives: Healthy individuals have significant beat-to-beat variability in heart rate, and this variability decreases with mental stress. We aim to use heart rate variability (HRV) to objectively compare mental stress levels in otologic surgeons at rest and during key portions of procedures. Design: Pilot study. Setting: Operating room and laboratory. Participants: Two neurotology fellows performed six mastoidectomy and facial nerve (FN) dissections in the operating room and six in a cadaver lab while continuous electrocardiograms were measured wirelessly. Five-minute samples were recorded during resting, preoperative, mastoidectomy, and FN dissection. Beat-to-beat time intervals were analyzed in time and frequency domains. The standard deviation of normal beat-to-beat intervals (SDNN) and the ratio of low frequency to high frequency power (LF/HF, measure of sympathetic tone) were calculated. Decreases in SDNN and increases in LF/HF indicate elevated mental stress. Results: Mean resting SDNN was 43.9 ± 9.2 ms, not statistically different from preoperative SDNN (34.1 ± 8.2 ms, p = 0.13). SDNN decreased during mastoidectomy (29.4 ± 11.7 ms) and FN dissection (22.8 ± 3.1 ms), which was significant compared to preoperative values (p = 0.03). Intraoperative LF/HF increased for FN dissection (6.8 ± 2.6) compared to resting (2.2 ± 0.7, p = 0.004), indicating increased sympathetic tone. Mastoid and FN cadaveric procedures resulted in SDNN of 33.6 ± 3.8 and 32.9 ± 4.7 ms, respectively, not statistically different from preoperative values (p = 0.82 and p = 0.94, respectively). Cadaveric FN dissection did not result in increased LF/HF (2.4 ± 0.9) compared to resting (p = 0.94). Conclusions: Decreased HRV and increased sympathetic tone were observed intraoperatively, indicating high levels of mental stress, particularly with FN dissection. Similar changes were not found during cadaveric dissections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the pandemic coronavirus disease 2019 (COVID-19), which has had a devastating impact on society. Here, we summarize proteomic research that has helped elucidate hallmark proteins associated with the disease with respect to both short- and long-term diagnosis and prognosis. Additionally, we review the highly variable humoral response associated with COVID-19 and the increased risk of autoimmunity.