Abstract
Pulmonary exacerbations (PEx) are clinically impactful events for
individuals with CF. Unfortunately, many CF individuals with PEx fail
to regain their baseline lung function despite treatment. The objective
of this study was to use unbiased proteomic technology to identify
novel blood protein biomarkers that change following intravenous (IV)
antibiotic treatment and to explore if changes correlate with clinical
response by the end of treatment. Blood samples from 25 PEx events
derived from 22 unique CF adults were collected within 24 hours of
hospital admission, day 5, day 10, and IV antibiotic completion.
Three-hundred and forty-six blood proteins were evaluated with
label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS)
quantitative proteomics and immunoassays. Forty-seven plasma proteins
changed significantly following 5 days of IV antibiotic treatment
(q-value ≤ 0.10). Early change in IGF2R from hospital admission to day
5 correlated with overall change in symptom score (CFRSD-CRISS) by the
end of treatment (r = −0.48, p-value = 0.04). Several plasma proteins
identified and quantified by label-free LC-MS/MS changed early
following treatment with IV antibiotics and many of these proteins are
involved in complement activation and inflammatory/immune-related
pathways. Early change in IGF2R correlated with symptom response
following IV antibiotic treatment and requires further validation as a
predictive biomarker of symptom response.
Subject terms: Predictive markers, Medical research
Introduction
Cystic fibrosis (CF) is a life-limiting autosomal recessive disease
affecting over 70,000 people worldwide^[36]1. Individuals with CF
experience recurrent pulmonary exacerbations (PEx) that are
characterized by intermittent worsening in respiratory signs and
symptoms^[37]2–[38]4 and are associated with increased morbidity and
irreversible loss in lung function^[39]5,[40]6. CF PEx are commonly
triggered by respiratory viruses, clonal shifts of colonizing bacteria,
and sometimes non-infectious causes (air pollution, medication
non-adherence) and are typically treated with antibiotics and increased
airway clearance therapies (i.e. hypertonic saline and dornase
alfa)^[41]2,[42]4. Based on data from the 2017 Cystic Fibrosis
Foundation Patient Registry, over 40% of CF adults were diagnosed with
at least one PEx that required treatment with intravenous (IV)
antibiotics^[43]7. Unfortunately, many individuals with CF fail to
regain their baseline lung function despite IV antibiotic
treatment^[44]8.
Suboptimal PEx outcomes may be due to delayed recognition and
treatment, widely varied treatment decisions, and differences in the
approach to monitoring treatment response, including recovery in lung
function and/or resolution in signs and symptoms^[45]9,[46]10. A novel
adjunctive strategy that can provide an additional dimension to the
objective monitoring of PEx treatment response is desired as it has the
potential to improve clinical outcomes for CF individuals.
Biomarkers that are measured objectively and reproducibly have been
studied in order to help guide therapeutic interventions in diseases
such as COPD^[47]11–[48]13. In CF, blood-based biomarkers that reflect
systemic inflammation, such as C-reactive protein and calprotectin,
decrease significantly following PEx treatment^[49]14–[50]18. However,
for a biomarker to aid in clinical decision making, an early change is
potentially more informative in assisting treatment decisions as it
provides the opportunity for CF physicians to modify treatments earlier
than if they had waited for the patient to respond or not clinically,
which can take longer in some patients. Based on the results of a prior
study performed by our group, admission but not early change in CRP was
found to be useful in predicting treatment outcomes and therefore CRP
does not appear to be a useful marker of early response to PEx
treatment in CF^[51]19.
In this study, we recruited adult CF subjects who were diagnosed with a
PEx and required hospitalization for IV antibiotic treatment. We
prospectively collected blood, symptom diaries, and spirometry (e.g.
FEV[1]) within 24 hours of admission, day 5, day 10, and IV antibiotic
treatment completion. Blood samples from these CF subjects have
previously been evaluated in a prior study using multiple-reaction
monitoring mass spectrometry (MRM-MS), a targeted proteomics
approach^[52]20. The objective of this study was to use label-free
(untargeted) liquid chromatography-tandem mass spectrometry (LC-MS/MS)
to identify novel blood protein biomarkers that are associated with
early response to IV antibiotics (i.e. from hospital admission to day
5) and to determine if early changes correlate with clinical outcome by
the end of IV antibiotic treatment, in terms of improvement in lung
function and symptoms.
Results
Clinical characteristics at hospital admission for PEx
A total of 25 PEx events from 22 unique CF subjects were eligible for
this study. Clinical characteristics of participating subjects at
hospital admission (V1) are summarized in Table [53]1. In brief, mean
baseline lung function was 63.4 (21.5) % predicted and 60% of CF
subjects had moderate to severe airflow obstruction (FEV[1 < ]70%
predicted). Mean FEV[1]% predicted and CF Respiratory Symptom
Diary-Chronic Respiratory Infection Symptom Score (CFRSD-CRISS) at V1
were 53.2 (SD 20.7) % predicted and 50.5 (SD 7.5), respectively. The
modified Fuchs exacerbation score ranged from 4 to 8 with a median
score of 6. Fourteen PEx events (56%) were characterized by a > 10%
relative drop in FEV[1]% predicted when compared to baseline. Over half
of PEx events (13/25, 52%) were non-acute with the patient describing a
change in symptoms for at least two weeks prior to hospitalization and
4 of the PEx events were characterized by the receipt of oral
antibiotics prior to hospitalization.
Table 1.
Clinical characteristics at hospital admission (V1).
Clinical characteristics
Number of PEx 25
Number of subjects 22
Age, mean (SD) 34.8 (12.9)
Female, No. (%) 12 (48)
Genotype, No. (%)
ΔF508 Homozygous 11 (50)
[MATH: Δ :MATH]
F508 Heterozygous 7 (32)
Other (non-
[MATH: Δ :MATH]
F508) 4 (18)
FEV[1]% predicted, mean (SD) 53.2 (20.7)
>10% relative drop FEV[1]% predicted from baseline, No. (%)^a 14 (56)
BMI, mean (SD) 21.7 (3.5)
CFRSD-CRISS, mean (SD) 50.5 (7.5)
Modified Fuchs Score, median (range) 6 (4 to 8)
Best FEV[1]% predicted in 6 months prior to PEx, mean (SD) 63.4 (21.5)
Best FEV[1]% predicted in 6 months prior to PEx, No. (%)
<40 4 (16)
40–69 11 (44)
70–89 4 (16)
≥90 6 (24)
Sputum Microbiology, No. (%)
P. aeruginosa 14 (56)
MSSA 12 (48)
MRSA 4 (16)
Burkholderia cepacia complex 3 (12)
Symptom Onset, No. (%)
>2 weeks 13 (52)
<2 weeks 12 (48)
[54]Open in a new tab
Abbreviation: PEx, pulmonary exacerbations; FEV[1], forced expiratory
volume in 1 second; BMI, body mass index; CFRSD-CRISS, CF Respiratory
Symptom Diary-Chronic Respiratory Infection Symptom Score; P.
aeruginosa, Pseudomonas aeruginosa; MSSA, Methicillin-sensitive S.
aureus; MRSA, Methicillin-resistant S. aureus.
^aBaseline lung function is defined as the best FEV1% predicted in the
6 months prior to the index PEx.
Clinical outcomes of PEx treatment
Clinical outcomes of PEx treatment are summarized in e-Table [55]1. For
25 PEx events, the median duration of IV antibiotic treatment was 14
days (ranged from 13 to 24 days). The FEV[1]% predicted increased from
V1 to each of the subsequent time points (V2, V3, and V4) but overall
changes were not significant (e-Table [56]1, Fig. [57]1a). Majority of
the PEx events (n = 21, 84%) recovered to 90% of baseline lung function
but fewer recovered to ≥100% of their baseline lung function (n = 9,
36%). Twenty out of 25 PEx events had symptom questionnaires completed
and the mean CFRSD-CRISS decreased significantly from V1 to each of the
following time points (V2, V3, V4; e-Table [58]1, Fig. [59]1b). Fifteen
(60%) events were characterized by >11-point decrease, which has been
defined as the minimum clinically important difference^[60]10,[61]21.
Figure 1.
[62]Figure 1
[63]Open in a new tab
Longitudinal changes in clinical outcomes and candidate blood proteins.
Abbreviation: PEx, pulmonary exacerbations; FEV[1], forced expiratory
volume in 1 second; CFRSD-CRISS, CF Respiratory Symptom Diary-Chronic
Respiratory Infection Symptom Score; IL-6, Interleukin-6; IL-8,
Interleukin-8; TNF-α, Tumor necrosis factor-α; SD, Standard deviation.
Statistical significance: p-value < 0.05 (*), p-value < 0.01(**),
p-value < 0.001(***).
Correlation between protein levels at hospital admission (V1) with clinical
and demographic factors
Serum IL-6 levels at hospital admission (V1) measured with immunoassay
inversely correlated with baseline FEV[1]% predicted (r = −0.40,
p-value = 0.046; e-Table [64]2). The levels of 16 plasma proteins
measured with LC-MS/MS also significantly correlated with baseline
FEV[1]% predicted at V1 (e-Table [65]2). Moreover, significant
correlations were identified between age and many plasma protein
abundances at V1 and the correlations varied by sex (e-Table [66]3).
Table 2.
Gene ontology (GO) biological process pathway enrichment analysis based
on DE proteins from V1 to V2.
GO term ID Term description Observed gene count Background gene count
q-value
GO:0002673 regulation of acute inflammatory response 11 92 2.51E-13
GO:0030449 regulation of complement activation 10 52 2.51E-13
GO:0070613 regulation of protein processing 12 116 2.51E-13
GO:2000257 regulation of protein activation cascade 10 54 2.51E-13
GO:0072376 protein activation cascade 10 74 1.28E-12
GO:0050727 regulation of inflammatory response 13 338 2.60E-10
GO:0002252 immune effector process 18 927 3.73E-10
GO:0006958 complement activation, classical pathway 7 34 9.93E-10
GO:0032101 regulation of response to external stimulus 16 732 1.28E-09
GO:0030162 regulation of proteolysis 16 742 1.43E-09
[67]Open in a new tab
Table 3.
Reactome pathway enrichment analysis based on differentially expressed
(DE) proteins from V1 to V2.
RCTM term ID Term description Observed gene count Background gene count
q-value
HSA-166658 Complement cascade 11 56 1.21E-15
HSA-977606 Regulation of Complement cascade 10 47 1.10E-14
HSA-168249 Innate Immune System 20 1012 4.56E-12
HSA-168256 Immune System 24 1925 7.71E-11
HSA-109582 Hemostasis 13 601 4.48E-08
HSA-114608 Platelet degranulation 8 125 4.48E-08
HSA-140877 Formation of Fibrin Clot (Clotting Cascade) 6 39 4.48E-08
HSA-76002 Platelet activation, signaling and aggregation 9 256 3.12E-07
HSA-166663 Initial triggering of complement 4 21 8.68E-06
HSA-140837 Intrinsic Pathway of Fibrin Clot Formation 4 22 9.31E-06
[68]Open in a new tab
Longitudinal changes in candidate and LC-MS/MS blood proteins
Serum IL-6 levels significantly decreased from hospital admission (V1)
to each of the following time points (V2, V3, V4; e-Table [69]1,
Fig. [70]1c), whereas significant changes were not identified between
subsequent time points, which is consistent with IL-6 changing early in
response to IV antibiotic therapy. Serum calprotectin levels
significantly decreased from hospital admission (V1) to treatment
completion (V4) but not at earlier time points (V2, V3). Significant
change in the levels of serum IL-8 and TNF-
[MATH: α :MATH]
were not identified between V1 and any of the subsequent time points.
Following adjustment for baseline lung function, sex, age, and the
interaction between sex and age, 47 proteins changed significantly from
V1 to V2 with a FDR cut-off q-value ≤ 0.10 (e-Table [71]4, Fig. [72]2a)
but just 6 proteins changed significantly from V1 and V4 with a FDR
cut-off q-value ≤ 0.10 (e-Table [73]5, Fig. [74]2b).
Figure 2.
[75]Figure 2
[76]Open in a new tab
Volcano plot demonstrating blood proteins measured with LC-MS/MS with
statistically significant fold-change from: (a) V1 to V2 and (b) V1 to
V4.
Protein-protein interaction (PPI) network and pathway enrichment analysis by
STRING algorithm
The online STRING database (version 11.0) was applied to identify the
protein-protein interaction (PPI) network using differentially
expressed (DE) proteins and their most enriched molecular pathways
following IV antibiotic treatment. The PPI network was constructed by
the STRING algorithm after analyzing 47 proteins that changed
significantly (q-value ≤ 0.10) between V1 and V2 (Fig. [77]3) and the
top 10 enriched GO biological process terms (Table [78]2) and Reactome
pathways (Table [79]3) are presented. Twenty-two of the 47 DE proteins
from V1 to V2 were involved in the following
immune/inflammatory-related GO biological processes: regulation of
complement activation, regulation of acute inflammatory response,
regulation of inflammatory response, immune effector process, and
complement activation/classical pathway (Table [80]2, e-Fig. [81]2).
Based on the Reactome database, 24 of the 47 DE proteins were involved
in 5 immune related pathways (Table [82]3, e-Fig. [83]3). Similar
analyses were not applied to DE proteins between V1 and V4 since only 6
proteins were identified.
Figure 3.
[84]Figure 3
[85]Open in a new tab
Protein association network based on differentially expressed (DE)
proteins from V1 and V2.
Correlation between early change in blood protein levels from hospital
admission (V1) to Day 5 (V2) with changes in clinical outcomes from hospital
admission (V1) to IV antibiotic completion (V4)
Among 47 DE proteins identified and quantified with LC-MS/MS, early
change (V1 to V2) in the levels of just one protein, insulin like
growth factor 2 receptor (IGF2R), inversely correlated with overall
change in CFRSD-CRISS from V1 to V4 (r = −0.48, p-value = 0.04;
e-Table [86]6). No significant correlations were identified between
early change in the levels of 47 DE proteins and relative change in
FEV[1]% predicted from V1 to V4. Additionally, early change in the
levels of candidate blood proteins did not correlate with overall
change in either FEV[1]% predicted or CFRSD-CRISS from V1 to V4.
A post-hoc sensitivity analysis indicated that the correlation between
early change in IGF2R with overall change in CFRSD-CRISS from V1 to V4
was generally consistent when we randomly selected one PEx from each of
the three subjects who had repeat PEx (e-Table [87]6).
Correlation between overall change in blood protein levels from hospital
admission (V1) to IV antibiotic completion (V4) with changes in clinical
outcomes from hospital admission (V1) to IV antibiotic completion (V4)
Six plasma proteins identified and measured with LC-MS/MS significantly
changed from V1 to V4 but no significant correlations with relative
change in FEV[1]% predicted or absolute change in CFRSD-CRISS from V1
to V4 were observed. Similarly, overall change in the levels of
candidate blood proteins did not correlate with overall change in
either FEV[1]% predicted or CFRSD-CRISS from V1 to V4.
Discussion
This is the first study to apply untargeted LC-MS/MS quantitative
proteomics to identify blood proteins that change in response to IV
antibiotics during the treatment of CF PEx. In addition to confirming
blood protein biomarkers previously reported to change following IV
antibiotics, including serum IL-6, calprotectin and plasma C-reactive
protein (CRP), several novel plasma proteins were also identified with
LC-MS/MS^[88]16,[89]22. Interestingly, more proteins exhibited changes
early during the treatment course (i.e. by day 5 of treatment) and
relatively fewer towards the end of the treatment. Based on pathway
enrichment analysis many of these proteins are involved in complement
activation and regulation of the inflammatory response/immunity.
Proteins that changed early and remained significant by the end of IV
antibiotic treatment included serum IL-6 and plasma CRP. However, early
and overall change in serum IL-6 and plasma CRP did not correlate with
changes in clinical outcomes, including FEV[1]% predicted and
CFRSD-CRISS scores, by the end of treatment. Calprotectin is a
candidate marker that also changed significantly by the end of
treatment but also did not correlate with changes in clinical outcomes.
In contrast, early change (V1 to V2) in insulin like growth factor 2
receptor (IGF2R; also known as cation-independent mannose-6-phosphate
receptor or CI-M6PR) levels significantly correlated with symptom
response (i.e. CFRSD-CRISS) by the end of IV antibiotic therapy. IGF2R
is a multi-functional binding protein capable of binding insulin growth
factor 2 (IGF2), mannose-6-phosphate (M6P), and retinoic acid^[90]23.
Depending on the ligand to which it binds, IGF2R is involved in
modulating a number of biological pathways including cell migration,
wound healing, angiogenesis, apoptosis and the response to viral
infection. Although it has not been studied in the context of CF
previously, it is also induced by inflammatory mediators and has been
studied as a candidate marker for systemic inflammation in other
patient populations such as HIV^[91]24.
Many of the proteins that were found to be downregulated by day 5 of
treatment are involved in complement activation (i.e. complement
proteins C1r, C1q, C4b, C4b-binding protein, C8, C9). The complement
proteins are key components of the innate immune system which promote
neutrophilic inflammation and defend against pathogens^[92]25.
Unregulated or persistent complement activation triggers a destructive
inflammatory cascade which may lead to lung tissue damage and cause
progressive loss of lung function^[93]25,[94]26. Elevated levels of
pro-inflammatory complement proteins have been observed in the sputum
of individuals with CF^[95]26. Despite the potential lung protective
effects of downregulating the complement system early during PEx
treatment, we did not observe a greater recovery of lung function in
such individuals but this study was small and therefore this warrants
further study.
This study has a number of important limitations. Only 25 PEx events
were analyzed in this exploratory study and therefore this study could
have been underpowered to examine relationships between biomarkers and
treatment outcomes. Furthermore, there was minimal decrease observed in
FEV[1]% predicted at the time of hospitalization in comparison to
stable baseline as only half of the PEx events were characterized by a
>10% relative drop in FEV[1]% predicted from baseline. As a result, the
improvements in FEV[1]% predicted in response to treatment were
relatively modest and may have limited the potential to identify
significant correlations with changes in blood proteins levels. As this
was an untargeted discovery study, a large number of proteins were
identified with LC-MS/MS proteomics and found to change significantly
following treatment but the multiple statistical comparisons performed
could have inflated the type 1 error. As such, the Benjamini-Hochberg
method was applied to adjust for multiple-testing with a cut-off
q-value of ≤0.10. However, this approach may have been too stringent
and resulted in false negatives at this discovery stage.
In conclusion, by using label-free LC-MS/MS quantitative proteomics, we
identified several blood proteins involved in complement activation and
inflammatory/immune-related pathways that changed in response to IV
antibiotic treatment. Early change in IGFR2 correlated with symptom
improvement by the end of treatment and requires further validation as
an early marker of symptomatic treatment response in individuals with
CF.
Methods
Study ethics
The research protocol was approved by the University of British
Columbia Providence Health Care Research Institute Research Ethics
Board (UBC-PHC REB number H12-00835). Informed written consent was
obtained from participating subjects. All methods were performed in
accordance with the relevant guidelines and regulations to protect
human subjects and ensure that participants remain de-identified during
samples analysis and data reporting.
Study cohort
Adult CF subjects were recruited prospectively when diagnosed with a
PEx and admitted to St. Paul’s Hospital (Vancouver, BC) for intravenous
(IV) antibiotic therapy between July 1, 2013 and June 30, 2015. The PEx
events were defined according to changes in respiratory symptoms with a
modified Fuchs PEx score of at least 4/12 requiring hospitalization for
IV antibiotic treatment^[96]3. Subjects were excluded if they had a
history of solid organ transplantation or were receiving chronic
immunosuppressive treatment. Participating subjects received IV
antibiotic treatment in conjunction with airway clearance therapies but
the IV antibiotic regimen and duration of PEx treatment were left to
the discretion of the most responsible CF physician.
Blood samples and clinical outcomes
Blood samples and clinical outcomes were obtained from subjects within
24 hours of hospitalization for IV antibiotic therapy (V1), treatment
day 5 (V2), treatment day 10 (V3), and IV antibiotic treatment
completion (V4). Blood samples (serum, EDTA-treated) were collected and
processed following standard operating procedures and then stored at
−80 °C until thawing for batched analysis.
Clinical characteristics including age, sex, baseline lung function
(i.e. FEV[1]% predicted), PEx requiring IV antibiotic treatment in the
prior year, sputum microbiology, were collected at hospital admission
(V1). Baseline lung function was defined as the best FEV[1]% predicted
in the 6 months prior to the index PEx. CF Respiratory Symptom
Diary-Chronic Respiratory Infection Symptom Score (CFRSD-CRISS) and
FEV[1]% predicted were recorded at V1 and each of the following time
points (V2, V3, V4). CFRSD-CRISS is scaled from 0 to 100 points with a
higher score indicating more severe respiratory symptoms^[97]21.
Clinical outcomes of interest included absolute change in CFRSD-CRISS
and relative change in FEV[1]% predicted from admission (V1) to
treatment completion (V4). Absolute change in FEV[1]% predicted was not
analyzed as it is largely influenced by baseline lung function with
larger increases seen with higher baseline lung function^[98]9,[99]10.
Untargeted proteomic profiling of blood proteins
Plasma samples were analyzed with label-free liquid
chromatography-tandem mass spectrometry (LC-MS/MS) at the University of
British Columbia Proteomics Core Facility. In brief, to facilitate the
analysis of less abundant plasma proteins, fourteen of the most highly
abundant proteins were first immunodepleted using the Human 14 Multiple
Affinity Removal Spin Cartridge (Agilent Technologies, Santa Clara,
CA), which removes albumin, immunoglobulin (Ig) G, alpha 1-antitrypsin,
IgA, transferrin, haptoglobin, fibrinogen, alpha 2-macroglobulin, alpha
1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII,
complement C3 and transthyretin^[100]27. Remaining plasma samples were
trypsin-digested overnight as previously described^[101]27. Resulting
peptides were desalted and purified with C-18 STop And Go Extraction
(STAGE) Tips^[102]28. Purified peptides were fractionated using the
Agilent 1100 HPLC system at 50 μL/min flow rate^[103]29. The analytical
column was operated at 50 °C using an in-house packed 75 μm C18 column
heater. The trap column that was added onto the analytical column was a
2 cm-long, 100 μm-inner diameter fused silica, packed with 5
μm-diameter Aqua C-18 beads (Phenomenex, Torrance, CA). Analytical
gradient was set at 75 minutes: changing from 10% to 35% Buffer B (80%
acetonitrile, 0.1% formic acid) for the first 60 minutes and then wash
with 100% Buffer B for 15 minutes. The sample was initially
fractionated into 96 wells (45 seconds per well) then pooled in a
noncontiguous manner (every 6th well was pooled) resulting into 6
fractions for further LC-MS/MS analysis^[104]29. Six fractions were
then loaded into the Impact II Q-ToF mass spectrometer (Bruker,
Germany)^[105]30. Peptides identified by LC-MS/MS were searched with
MaxQuant software (version 1.5.3.30) with default
label-free-quantitation setting and match-between runs options
enabled^[106]31. All plasma samples were evaluated in duplicate and
mean values were used for analyses. The LC-MS/MS data were deposited in
the PRoteomics IDEntifications (PRIDE) database under accession number
PXD016089.
Analysis of candidate blood proteins
Five low-abundance candidate blood proteins, including interleukin
(IL)-
[MATH: β :MATH]
, IL-6, IL-8, tumor necrosis factor (TNF)-
[MATH: α :MATH]
, and calprotectin, that are beyond the detection limits of LC-MS/MS
were analyzed in serum samples with multiplex electrochemiluminescence
immunoassays (Meso Scale Discovery, Carlsbad, CA). Among these five
low-abundance blood proteins, IL-1
[MATH: β :MATH]
was below the detection limits for most of the samples, and therefore,
was excluded from subsequent analyses. All assays were performed in
duplicate with mean coefficient of variation (CV) < 5% and mean values
were used for analyses.
Protein-protein interaction (PPI) network and pathway enrichment analysis
The differentially expressed (DE) proteins identified by LC-MS/MS were
applied as inputs for PPI network and pathway enrichment analysis. The
STRING database (version 11.0) was utilized to assess the protein
functional association^[107]32. The active protein-protein interactions
were identified based on experimentally determined interactions and
curated databases, such as Gene ontology, KEGG, Reactome databases. The
predicted protein-protein interactions included gene neighborhood, gene
fusion, gene co-occurrence, text-mining, co-expression, and protein
homology^[108]32. Enriched Gene Ontology (GO) biological process term
and Reactome pathways were reported and proteins involved in the immune
and inflammation related pathways were highlighted in the constructed
PPI network.
Statistical analyses
Statistical analyses were performed using R (version 3.5.0, the R
Foundation for Statistical Computing, Vienna, Austria) and Prism 8
(GraphPad, La Jolla, CA). Continuous variables were presented as
mean ± standard deviation (SD). Categorical variables were reported as
number with proportions. Longitudinal changes in the levels of
candidate blood proteins and changes in clinical outcomes (i.e.,
FEV[1]% predicted and CFRSD-CRISS) were analyzed with non-parametric
Kruskal-Wallis test followed by the post-hoc Dunn’s test to correct for
multiple comparisons.
LC-MS/MS data was pre-processed as described in the flow diagram
(e-Fig. [109]1). Proteins were excluded if they were identified as
reversed and/or contaminated by MaxQuant software during peptide
searching or detected in less than 25% of blood samples. Three-hundred
and forty-one proteins passed the quality control matrix. Missing
values from these 341 proteins were imputed with half of the minimum
abundance of each protein across the analyzed samples and then, the
levels of proteins were log2 transformed before subsequent analyses.
Fold-changes of blood proteins from V1 to V2 and V1 to V4 measured with
LC-MS/MS were analyzed with the limma R software package and adjusted
for baseline lung function, sex, age, and an interaction term between
sex and age. The Benjamini-Hochberg method was applied to correct for
multiple comparisons and false discovery rate (FDR) adjusted p-values
(q-value) ≤ 0.10 were reported for differentially expressed (DE)
proteins.
To assess how blood protein levels at V1 might be confounded by
baseline disease severity and demographic factors, correlation between
blood protein levels at V1 and age, sex, and baseline lung function
were evaluated with Spearman’s correlation. Additionally, correlations
between early change (V1 to V2) and overall change (V1 to V4) in blood
proteins with relative change in FEV[1]% predicted and absolute change
in CFRSD-CRISS from V1 to V4 were examined with Spearman’s correlation.
Statistical significance was reported when two-sided p-values were
≤0.05.
To ensure our findings were robust, we performed a post-hoc sensitivity
analysis that randomly selected one PEx from three subjects who had
repeat PEx.
Supplementary information
[110]Dataset 3^ (15.3MB, zip)
[111]Supplementary materials^ (2.5MB, pdf)
[112]Dataset 1^ (73.9KB, xlsx)
[113]Dataset 2^ (9.9KB, txt)
Acknowledgements