There is an urgent need for increased capacity of accurate quantification of proteins to better understand health and disease. Mechanistically, higher throughput is needed to cope with biological variation; clinically the COVID-19 pandemic has underscored the critical demand for high throughput methods for measuring e.g. acute phase response (APR) proteins or the SARS-CoV-2 viral proteins to characterize infection dynamics. Conventional protein biomarker screening approaches generally rely on capture reagents with colorimetric detection strategies (e.g., immunoassays) which are parallelized to allow for rapid throughput. An alternative detection strategy is mass spectrometry coupled with liquid chromatography (LC-MS). However, this is done sequentially, challenging throughput. Acoustic ejection mass spectrometry (AEMS) has emerged as a potential paradigm shift for ultra-fast screening by MS, at throughputs as fast as 1-2 seconds per analyte per sample. Here, we present the first quantitative AEMS assays utilizing peptide immunocapture to enrich 10 APR protein markers from plasma. We quantified 10 proteins from 267 plasma samples in triplicate in 4.8 hours with %CV between 4.2 and 10.5%, representing a 20-fold speed increase over LC-MS. Similarly, we show the capability of direct SARS-CoV-2 peptide quantification in nasopharyngeal swabs. By leveraging the speed of AEMS technology, combined with the enrichment and selectivity provided by peptide immunocapture, this generalizable approach enables both biomarker screening and diagnostic detection on very large cohorts, accelerating our ability to translate biological discoveries into future clinical use.

Sample preparation APR-panel: The sample preparation workflow described by Razavi et al. (2016) was altered to make it compatible with the Biomek i7 automated workstation (Beckman-Coulter, CA, USA). SARS-CoV-2: Sample preparation was performed as described by Van Puyvelde et al. (2022). Finally, 5 µL of a SIL mixture (55 fmol/µL) of the three NCAP peptide targets (ADETQALPQR, AYNVTQAFGR and KQQTVTLLPAADLDDFSK) was added before peptide enrichment Prior to the addition of the antibody-coupled magnetic bead immunoadsorbents, a step was included to fully resuspend the beads. To perform the SARS-CoV-2 peptide detection assay, magnetic beads coupled with monoclonal antibodies for three specific peptides for the NCAP protein, namely AYNVTQAFGR, ADETQALPQR, and KQQTVTLLPAADLDDFSK were combined in equal volumes. Next, 30 μL of this mixture was added to the nasopharyngeal swab samples after trypsin digestion. For the APR panel, 20 µL of the magnetic bead mixture coupled with monoclonal antibodies for the 10 target peptides (Supp. table 1) was added to the digested plasma samples. After antibody-bead addition, washing and elution was performed as previously described, however a different wash buffer was used (10 mM ammonium bicarbonate (ABC), 5% MeOH, 0.00025% CHAPS) and one additional bead washing step was included ​(16)​. Prior to AEMS analysis, the samples containing the purified peptides were transferred to an Echo qualified 384-well plate, spun at 1000g for 1 minute to achieve a uniform fluid meniscus. For the nasopharyngeal swabs, an additional Solid-Phase Extraction (SPE) step was included to further clean up the samples, using the HLB µElution kit. AEMS analysis Samples were analyzed in triplicate at 1-3 seconds per sample from a 384-well plate using MRM analysis on an Echo MS system with the SCIEX Triple Quad 6500+ mass spectrometer (SCIEX). AE conditions were set as following: Using a carrier solvent of 80% acetonitrile/200 nM medronic acid at a flow rate of 500 uL/min, with a droplet count of 100 – 300 nL total ejection volume. The source conditions were set as following: Gas1 = 90, Gas2 = 70, CurtainGas = 25, SourceTemp = 400°C, IonSpray voltage = 5000V. Two MRM transitions (dwell time of 10 ms each) were monitored per peptide, both for the heavy and light peptides (Supplementary Table 2 and 3) plus an extra MRM to monitor CHAPS (dwell time 3ms) in the elution buffer which served as the Marker well, required for successful file splitting LC-MS analysis An Eksigent NanoLC 425 System (SCIEX, CA) plumbed for microflow chromatography was used and operated in direct injection mode with the SCIEX Triple Quad 6500+ mass spectrometer. The column used was a 50x0.3mm Kinetex XB-C18 Column (2.6 µm, 100 Å) and the temperature was controlled at 30°C. A 2.5 min gradient from 5-27% mobile phase B was used for elution of peptides, then two rapid washes were performed for a total LC run time of 6.5 mins (see Supplementary Table 4). Sample injection protocol was 2 mins for a total sample analysis time of 8.5 mins. For the inflammation panel samples, 1 µL of the final sample was injected. For the SARS-CoV-2 sample, the final sample was diluted ¼ with Mobile phase A and 2 µL was injected on the LC-MS. The same two MRM transitions that were used for Echo MS analysis were used for LC-MS analysis, run using the Scheduled MRM algorithm (Supplementary Table 2). The source conditions were set as follows: Gas1 = 20, Gas2 = 20, CurtainGas = 35, SourceTemp = 100C, IonSpray voltage = 5000V.

Remnant plasma samples from the Cedars-Sinai Medical Center (CSMC) Biobank Resource, from whole blood collected in anticoagulant-treated (EDTA) tubes, were obtained from a single-center observational study of adult subjects admitted to either the medical floor or the intensive care unit (ICU) of CSMC between March 23 and May 10, 2020, and who were experiencing symptoms related to Covid-19. The samples selected for this study were collected within the first 1-2 days from admittance. Ethical approval was obtained from the Institutional Review Board on Research Involving Human Subjects (CSMC IRB#: STUDY00000621). In addition, gender pooled (100 females and 100 males) EDTA human plasma from healthy individuals (abbreviated as JVE Plasma) was purchased from BiolVT (Westbury NY, USA). Residual Covid-19 nasopharyngeal samples were obtained from the AZ Delta Hospital, Roeselare, Belgium, with approval of the University Hospital Ghent ethics committee (BC-09263). Both studies were performed in accordance with the Helsinki declaration.