Fig 1: Interaction of CRTAC1 and CFP. (a) CFP concentrations predicted by ratio of IBAQ values with ALB in the hospitalized patients divided into groups without or with COVID‐19 and further divided into patients who were not or were in the intensive care unit at time of enrollment, as in Figure 2a. For comparisons of groups: ***p ≤ 0.001, **p ≤ 0.005 (Tukey's multiple comparisons posttest). (b) Scatter plot of nM concentrations of CFP predicted by ratio of IBAQ values with ALB and CRTAC1 determined by ELISA. Pearson correlation coefficient (r) for COVID‐19 (closed circles, solid line) = 0.37, p = 0.0001; for non‐COVID‐19 (open circles, dashed line) r = 0.26, p = 0.22. (c) Binding of 140 nM CRTAC1 (10 μg/mL) to immobilized CFP (coated at 200 nM [10 μg/mL]) as detected with anti‐CRTAC1. (d) Binding as detected with anti‐CRTAC1 of increasing concentrations of CRTAC1 in the absence or presence of 1 mM CaCl2 to immobilized CFP coated at 200 nM in TBS. (e) Inhibition by preincubation with 200 nM soluble CFP of binding of 43 nM CRTAC1 (3 μg/mL) to CFP coated at 60 nM (3 μg/mL); bound CRTAC1 detected with anti‐CRTAC1. Mean and standard error of the mean (SEM) of triplicate (c and e) or duplicate (d) wells.
Fig 2: Plasma CRTAC1 concentrations determined by ELISA in different subject groups and relation to hospital day and clinical severity score. (a) The 20 healthy control subjects, 55 patients with COPD, and 128 hospitalized patients divided into groups without (n = 26, non‐COVID, open circles) or with (n = 102, COVID, closed circles) COVID‐19 and further divided into patients who were not (n = 10 + 51, respectively, non‐ICU) or were (n = 16 + 51, respectively, ICU) in the intensive care unit at time of enrollment. ***p ≤ 0.001, *p ≤ 0.02 (t‐test for pairwise comparison of COPD versus healthy, otherwise Tukey's multiple comparisons posttest). (b) CRTAC1 concentration versus day of hospitalization. The 128 patients divided into groups without (n = 26, open circles, dashed line) or with COVID‐19 (n = 102, closed circles, solid line). Spearman rank correlation coefficient (r s) for COVID‐19 = −0.10, p = 0.30; for non‐COVID‐19 r s = −0.28, p = 0.16, for all r s = −0.29, p = 0.0009. (c) CRTAC1 concentration in another set of hospitalized patients with COVID‐19 (n = 5) who were sampled more than once with 3‐day intervals. D: CRTAC1 concentration versus APACHE (acute physiological assessment and chronic health evaluation) II score. The 75 patients given an APACHEII score are divided into groups without (n = 17, open circles, dashed line) or with COVID‐19 (n = 58, closed circles, solid line). r s for COVID‐19 = −0.33, p = 0.01; for non‐COVID‐19 r s = −0.20, p = 0.44; for all r s = −0.30, p = 0.009. Band, range of healthy subjects (17.2–59.7 nM).
Fig 3: Plasma CRTAC1 concentrations determined by ELISA in patients after COVID‐19 treated in or outside the hospital. (a) A subset (n = 16) of the 102 patients hospitalized with COVID‐19 were sampled 1 year after hospitalization, p < 0.0001 for a year later (1 year) versus the time of hospitalization (initial) (paired t test of log10 data). (b) Patients with long COVID (n = 127); p = 0.02 for long COVID versus healthy and p = 0.008 for long COVID versus COPD (t‐test). (c) Plot of plasma CRTAC1 versus age in long COVID patients without (n = 111, closed circles, solid line) and with (n = 16, open circles, dashed line) COPD, with linear regressions. (d) Plot of plasma CRTAC1 versus age in healthy controls (n = 20) with linear regression. Band, range of healthy subjects (17.2–59.7 nM).
Fig 4: Western blot and development of ELISA for CRTAC1 and comparison of CRTAC1 concentrations in plasma as determined by ELISA and mass spectrometry. (a) Left: SDS‐PAGE Gelcode Blue staining of recombinant human CRTAC1 (2 μg/well) under reducing conditions; right: immunoblots under reducing conditions using sheep polyclonal antibodies or mouse mAb to recognize recombinant human CRTAC1 (10 ng) (lane 1) or CRTAC1 in four plasma samples (lanes 2–5) estimated to vary in amount of CRTAC1 by ELISA as indicated below the lane. Positions of molecular size markers (kDa) on the left. (b) Representative ELISA for human CRTAC1: optical density (OD) at 450 nm versus a standard (std) curve of recombinant human CRTAC1 in nM or dilutions of three plasma samples for which the indicated concentrations were determined. (c) CRTAC1 concentration (log nM) of plasma samples from hospitalized patients with or without COVID‐19 determined by ELISA versus concentration predicted by mass spectrometry (MS) compared to ALB as described in the text. Shown are the linear regression of the log10 values and 95% confidence intervals; closed circles, samples with non‐imputed MS data; open circles, samples with imputed MS data, n = 128 of which 27 had imputed MS data. Pearson correlation coefficient (r) for all samples = 0.58, probability (p) < 0.0001; for non‐imputed samples r = 0.58, p < 0.0001; and for imputed samples r = 0.16, p = 0.42.
Fig 5: IBAQ scores and CVs of plasma proteins in hospitalized COVID‐19 patients. (a) Scatter plot of means of log10 IBAQ value versus coefficients of variation (CVs) calculated based on log10 IBAQ scores of the 501 proteins. Closed circles, secreted proteins; open circles, non‐secreted proteins. The proteins with the highest and lowest IBAQ scores (ALB and SPTA1, respectively) and one outlier (ASS1) are indicated. (b) Box plots (boxes representing medians and quartiles, whiskers representing minimum and maximum) of means of log10 IBAQ scores (left) and CVs (right) of secreted and not secreted proteins, p < 0.0001 for each comparison. (c) Pearson correlation coefficient (r) between log10 CRTAC1 determined by ELISA and log10 IBAQ value by mass spectrometry (MS) of the 501 plasma proteins. p for r outside the band <0.05.
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