Coronavirus infectious disease 19 was first reported in Wuhan, 47 overlaid with 2 µl of CHCA matrix and allowed to air-drying. The analysis of 126 each sample was conducted in triplicate. 127 128
Measurements were performed on an Autoflex III MALDI-TOF mass 130 spectrometer (Bruker Daltonics, Leipzig, Germany) equipped with a 200 Hz 131 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 3, 2020. ; https://doi.org /10.1101 /10. /2020 baseline correction, and intensity normalization were performed on each mass 143 spectrum. The average spectrum from the triplicates was obtained. Peaks were 144 detected and binned across all average spectra with a signal to noise ratio of 5 145 and a tolerance of 0.002. Peaks presents in less than 25% of the spectra were 146 rejected. All spectra from the groups under study were pre-processed, and peak 147 detection was applied to obtain an intensity matrix. The resulting matrix of peak 148 intensities was used for Principal Component Analysis (PCA) (9) and Machine 149 learning (ML) approaches (10) . To set up ML, the radial basis function was used 150 as the kernel function and ANOVA was used to select the 45 most relevant 151 peaks. 152 153
Three microliters of serum were diluted to 1 ml with 50 mM Ammonium 155 bicarbonate (0.2 µg/µl taking the average of plasma proteins as 80 mg/ml). One 156 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 3, 2020. ; https://doi.org /10.1101 /10. /2020 hundred µL of the dilution were reduced with 11 µl of 50 mM dithiothreitol for 30 157 minutes at 56°C and were alkylated with 12.5 µl of indole-3-acetic acid 20 mM 158 for 20 minutes in the dark at 37 °C. Total volume 123.5 µl, containing likely 24 overnight. Ten microliters of formic acid (FA) 5% were added to stop the 161 digestion. while non-severe were those without oxygen support (mild patients). Seventy-223 five percent of the samples from each group were randomly selected to form 224 the training set, while the remaining 25% of the samples were used for 225 validation as an independent test cohort. This process was repeated five 137) classified as clinically severe (grade 2) when the samples were collected, 248 which 48-96 h later evolved to critical (grade 3), were clearly clustered in the 249 group of critical patients using machine learning, while the remaining 8 patients 250 (70, 73, 66, 67, 62, 59, 53, 58) did not worsen to grade 3.
In this study we demonstrate that the molecular changes that occur in the sera 274
peptidome profiles that may be used as clinical classifiers. In addition, we show 276 that it is possible to predict the progression of the disease using the peptidome 277 signatures obtained with this technology. Finally, we provide strong evidences 278 that serum amyloid A2 protein is one of the major biomarkers of severe COVID-279 19 disease. 280
To our knowledge, only two previous studies reported the use of mass 281 spectrometry analysis of serum from COVID-19 patients to classify disease 282 severity (4, 5). However, both studies were performed using sophisticated 283 technologies, which are not available in most of the hospitals. Our challenge 284 was to test whether MALDI-TOF MS analysis, a simpler technology available in 285 most of the clinical microbiology laboratories for identification of microbial 286 species, was able to achieve similar results. Our study classified severe 287 patients with a high accuracy (90%), very similar to that obtained in a previous 288 report (93%) (4), with a very low number of false results. 289
We represented the changes of those peaks that exhibited major changes upon 290 grouping the patients according to CDC Chinese severity criteria, ranging from 291 scale 1 to scale 3 in a heatmap, which graphically illustrated how level changes 292 in these peaks reflected a progression from mild to critical Interestingly, our peptidome profile data identify the most important changes 294 within the severe patients, upon which a patient is put on oxygen supply. This 295 observation is consistent with the proteome analysis conducted by Messner et 296 al, who found that at molecular level the requirement of oxygen supply 297 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 3, 2020. ; https://doi.org /10.1101 /10. /2020 Islas Baleares (IdISBa). 349 350 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 3, 2020. ; https://doi.org /10.1101 /10. /2020 preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in
The copyright holder for this this version posted November 3, 2020. ; https://doi.org /10.1101 /10. /2020 
All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 3, 2020. ; https://doi.org /10.1101 /10. /2020 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted November 3, 2020. ; https://doi.org /10.1101 /10. /2020 