Mass Spectrometry-Based Proteomics Analysis of Nasal Fluid Uncovers Differentially Expressed Proteins as Potential Biomarkers of Allergic Asthma

To the Editor,

Asthma is a complex and varied disease characterised by episodic, reversible bronchoconstriction. However, current clinical biomarkers such as blood eosinophils, serum IgE, and exhaled nitric oxide are often insufficient to distinguish between different asthma sub-endotypes and do not reliably correlate with the disease’s severity [1]. These biomarkers are somewhat effective in identifying type 2-high asthma; however, they lack specificity and do not help in characterising type 2-low asthma. Nasal mucosal fluid could be a valuable source of asthma-related biomarkers because it directly reflects the respiratory tract environment. Although previous studies have explored nasal fluid proteomics to identify potential proxies for the lower airway, these approaches are not yet incorporated into routine diagnostics [2, 3] We conducted this exploratory pilot study to assess whether an unbiased mass spectrometric analysis of all detectable proteins in nasal fluid could differentiate between patients with allergic asthma and those with allergic non-asthma. We hypothesise that nasal fluid contains a wide array of inflammatory markers, offering a comprehensive view of various asthma endotypes and sub-endotypes.

Adult patients with mild-to-moderate asthma and allergic rhinitis (n = 9), as well as non-asthmatic controls with allergic rhinitis (n = 9), were recruited from the Brigham and Women’s Hospital (Boston, MA) Allergy Clinics. The study was approved by the local Institutional Review Board, and all participants provided written informed consent. All subjects tested positive to multiple environmental allergens through skin testing, with ages ranging from 25 to 74 years. None of the participants had used biologics, systemic corticosteroids, or intranasal steroids in the 5 days prior to nasal fluid collection. All asthmatic patients were on daily low-to-medium dose inhaled corticosteroids and classified as GINA Step 2–3.

Participants were mostly female (11 out of 18) and identified as White (13 out of 18). The sample also included three Hispanic, one Asian, and one Black or African American individual. There were no significant differences in age, sex, or race between the case and control groups. Nasal fluid was collected during a single visit between September and November 2023, using Nasosorption FX (Mucosal Diagnostics), a non-invasive, absorptive matrix strip designed to collect mucosal lining fluid. The strip was inserted into the anterior nostril for 1 min, then placed in 300 μL of phosphate-buffered saline and stored at −80°C. The nasal fluid was then pre-processed with an S-Trap mini column (Protifi CO2-mini). Peptides were labelled using the TMTpro 18-plex kit (Thermo Scientific), pooled, dried, and fractionated into 20 fractions via high-pH reversed-phase spin columns. These peptides were separated on a C18 column and analysed by liquid chromatography–tandem mass spectrometry, using an Orbitrap-Exploris 240 mass spectrometer (Thermo Scientific) coupled with an EASY-nLC 1200 nanoLC system. Raw mass spectrometry data files were processed with Proteome Discoverer (V3.0), utilising the Sequest HT and Chimerys (V1.8) search engines. Spectra were searched against the UniProt human proteome database (2019), with enzyme specificity set to trypsin and a maximum of two missed cleavages. The false discovery rates for peptides and proteins were both set at 1%.

Quantitative data were processed using Wyss Analysis Software for Proteomics (Lewandowski et al.). Proteins were considered differentially expressed if they met two criteria: an uncorrected Mann–Whitney U test P-value less than 0.05 and a fold change greater than 1.25 or less than 0.75 between asthma cases and controls. Since this study was exploratory and hypothesis-generating, the statistical analysis was not adjusted for multiple comparisons.

The mass spectrometric analysis identified a total of 2194 proteins in the nasal fluid samples. Among these, 32 proteins showed increased expression levels, while 13 exhibited decreased expression in the asthma group compared to the rhinitis-only control group (see Table 1).

TABLE 1 |.

Proteins over- and under-expressed in the nasal fluid of asthmatic vs. rhinitis-only controls.

Protein name	Gene symbol	Fold difference	Mann–whitney p
Enolase 3	ENO3	2.172	0.04
Proteasome 20S subunit beta 9	PSMB9	1.733	0.0142
Adducin 1	ADD1	1.683	0.0244
Carbohydrate sulfotransferase 5	CHST5	1.671	0.0106
Piwi like RNA-mediated gene silencing 2	PIWIL2	1.63	0.0028
Adaptor related protein complex 3 subunit delta 1	AP3D1	1.571	0.0078
Adenosine monophosphate deaminase 2	AMPD2	1.534	0.0142
RAB11-binding protein RELCH	RELCH	1.529	0.0106
Immunoglobulin heavy variable 1–2	IGHV1-2	1.491	0.0244
RNA 2′,3′-cyclic phosphate and 5′-OH ligase	RTCB	1.446	0.0188
Endonuclease, poly (U) specific	ENDOU	1.435	0.0188
Syndecan binding protein	SDCBP	1.428	0.0078
Acid phosphatase 1–2	ACP1-2	1.416	0.04
Fc gamma binding protein	FCGBP	1.368	0.0244
Taxi binding protein 3	TAX1BP3	1.348	0.0106
Thimet oligopeptidase 1	THOP1	1.338	0.0019
GTP binding protein 1	GTPBP1	1.335	0.0244
Serpin family A member 3	SERPINA	1.332	0.0315
Acetylserotonin O-methyltransferase like	ASMTL	1.327	0.0188
EPS8 signalling adaptor L1	EPS8L1	1.325	0.04
Glycosylphosphatidylinositol spec phospholipase D1	GPLD1	1.324	0.0188
Serpin family A member 1	SERPINA	1.322	0.0106
Alpha 2-HS glycoprotein	AHSG	1.31	0.0315
Complement factor I	CFI	1.308	0.0019
FRAS1 related extracellular matrix 3	FREM3	1.302	0.0142
Importin 5	IPO5	1.297	0.04
Carboxylesterase 1	CES1	1.297	0.0078
Matrix metallopeptidase 25	MMP25	1.297	0.0078
Retinol binding protein 4	RBP4	1.29	0.04
Serpin family A member 6	SERPINA	1.271	0.04
Mucin 16, cell surface associated	MUC16	1.26	0.0056
Methionyl aminopeptidase 1	METAP1	1.26	0.0078
Apolipoprotein C1	APOC1	0.749	0.04
Bridging integrator 2	BIN2	0.747	0.0056
Phosphatidylinositol binding clathrin assembly protein	PICALM	0.74	0.0315
Ribonuclease H2 subunit B	RNASEH2B	0.724	0.0188
Proline rich coiled-coil 2C	PRRC2C	0.723	0.0188
Heterogeneous nuclear ribonucleoprotein M	HNRNPM	0.718	0.0056
Epidermal growth factor rec path substrate 15 like 1	EPS15L1	0.712	0.0244
Nuclear autoantigenic sperm protein	NASP	0.685	0.04
Proliferation and apoptosis adaptor protein 15	PEA15	0.682	0.0142
High mobility group nucleosomal binding domain 3	HMGN3	0.678	0.0056
Ribosomal protein L28	RPL28	0.632	0.0188
Nudix hydrolase 1	NUDT1	0.552	0.04
RNA-splicing factor	CWC25-2	0.53	0.0056

Note: Proteins listed had a fold difference of > 1.25 or < 0.75 between the two patient cohorts, with a Mann–Whitney p value < 0.05.

Among the proteins upregulated in asthmatics, several may be of clinical relevance or have been previously associated with airway inflammation. Additionally, multiple members of the serpin family of serine protease inhibitors show differential expression across various chronic lung conditions [4]. Three serpin family proteins were found at higher levels in the nasal fluid of our asthmatic patients compared to controls. Notably, SERPINA3, also known as alpha-1-antichymotrypsin, showed a fold increase of 1.33 (p = 0.032). This protein inhibits neutrophil cathepsin G and is elevated in the plasma of patients with severe asthma compared to healthy individuals [5]. The nasal levels of the large mucin MUC16 increased by a fold difference of 1.26 (p = 0.006). This molecule may have immunologic significance, as it collaborates with TGFβ-1 to promote lung fibrosis and serves as a biomarker for disease progression in idiopathic pulmonary fibrosis [6]. A recent proteomics study of nasal lavage fluid found that SERPINB7 levels were elevated in the nasal fluid of asthmatic patients compared to non-asthmatics. Additionally, MUC16 levels were differentially expressed between patients with controlled and uncontrolled asthma [3]. The metalloproteinase MMP25, also known as leukolysin, was observed to be elevated in the nasal fluid of the studied asthmatic cohort, with a fold change of 1.30 (p = 0.008). MMP25 facilitates airway remodelling through the activation of MMP9, an enzyme responsible for basement membrane degradation. Furthermore, MMP25 has been linked to allergic conditions such as asthma and is found at higher levels in the exhaled breath condensate of asthmatics relative to healthy controls. Its levels also exhibit an inverse relationship with pulmonary function [7].

In our asthmatic cohort, certain anti-inflammatory proteins were downregulated in nasal fluid. For instance, apolipoprotein C1 (fold-difference x0.75, p = 0.040), which promotes a pro-resolving macrophage phenotype that protects against airway remodelling in asthma, showed decreased levels [8]. Additionally, reduced amounts of the nuclear autoantigenic sperm protein NASP (fold-difference x0.69, p = 0.040) suggest a potential immunologic mechanism. Mouse models of allergic airway inflammation have demonstrated that NASP plays a regulatory role in asthma by inhibiting mucus production [9].

This pilot study utilised unbiased mass spectrometry-based proteomic analysis of non-invasively collected nasal mucosal fluid. It identified differences in the nasal fluid proteome between patients with allergic asthma and non-asthmatic controls with allergic rhinitis. Although limited by a small sample size, the findings provide preliminary data to guide larger, more comprehensive investigations of nasal fluid proteomics in asthma endotyping. Future research should include asthma cohorts with varying disease severities and control levels, as well as different underlying asthma endotypes, to better explore the potential of nasal proteomics in these contexts.

Summary.

• The existing biomarkers for endotyping asthma are insufficient to fully identify the different asthma subendotypes.

• Examining proteomic differences in nasal fluid may provide a non-invasive biomarker to better endotype asthma.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.