Caution in Using Proteins Extracted from Exhaled Breath Condensate of Intubated Patients for Pediatric ARDS Studies

Objective We sought to analyze the quality of proteins extracted from the humidied moisture exchange lter (HMEF) of pediatric acute respiratory distress syndrome (PARDS) patients undergoing invasive mechanical ventilation. Results Proteins were extracted from ten lters from one control and six PARDS subjects. Between 0.69-1.69 mg of protein was extracted from each. Silver stain of these extracts identied only one discrete band compared to many discrete bands in mouse BALF. Liquid chromatography- mass spectrometry of this band and a corresponding band in mouse bronchoalveolar lavage uid identied them as human and mouse albumin respectively. Multiple other non-degraded proteins were obvious in mouse bronchoalveolar lavage uid but not proteins extracted from HMEFs.


Introduction
The identi cation of subtypes and endotypes in critical care medicine in general and acute respiratory distress syndrome (ARDS) in particular has generated hope that the area of personalized medicine in critical care may be dawning. To date, subtypes and endotypes in ARDS have been de ned by injury mechanism, physiologic parameters, and serum biomarkers (1). Serum biomarker identi cation of hypoand hyper-in ammatory subtypes have been well described (2; 3). Three prior publications have reported proteomic analysis of exhaled breath condensate in mechanically ventilated adults (4; 5; 6) indicating that direct proteomic assessment of the lower airways in ARDS may be possible.

Methods
We sought to determine the feasibility of analyzing proteins extracted from the humidi ed moisture exchange (HME) lters (Pall Corp, #1602292) of intubated pediatric ARDS patients after 12 hours of use (Cincinnati Children's Hospital Institutional Review Board Approval #2017 − 1345). After cutting open the casing, the cellulose lter material was extracted from 10 HME lters from 6 PARDS and 1 control subjects. The lter material was placed in the top compartment of a 15 kDa centrifugal lter unit (Millipore, UFC900308) and 10 mL of PBST was added to extract and concentrate proteins in exhaled breath condensate.

Results
The total protein extracted varied from 0.1 to 1.7 mg with a noticeable discoloration of ARDS extracts compared to control (Fig. 1A). However, silver stain of these specimens (Bio-Rad, Hercules, CA) identi ed only weak ~ 65 kDa bands. As a positive control, these bands were compared to C57BL/6 bronchoalveolar lavage uid with total protein extracted being 0.1 mg (Fig. 1B). Human and mouse 65 kDa bands were analyzed by MALDI-TOF and were identi ed as human and murine albumin respectively. Densitometry of discrete bands vs. total lane intensity as an estimate of intact, high-abundance proteins demonstrated 4-5% intact protein for HME ventilator specimens vs. 37% for mouse BAL uid. We concluded that, at least in our hands, the proteins extracted from the HME lters of intubated PARDS subjects were substantially degraded.

Discussion
Despite there being at least three previous studies assessing the protein in exhaled breath condensate of ARDS subjects, to our knowledge, this study is the rst to assess whether extracted proteins are intact by The human airway is an environment rich in proteases and other defense-related proteins, and it is perhaps not surprising that proteins collected from exhaled breath condensate would show a high degree of degradation. Most quantitative assays are antibody based, and depending on assay characteristics (polyclonal vs. monoclonal capture and detection antibodies, speci c assay chemistry, etc.) low levels of degradation might arti cially increase and higher levels of degradation arti cially decrease the amount of analyte quanti ed. Perhaps more importantly, differences in degradation might confound comparisons between samples. These differences could be due factors such as patient disease status, lter type, storage conditions, and protein extraction methods, none of which were examined in this study.
Nonetheless, our ndings indicate that protein degradation can be a substantial problem in studies of exhaled breath condensate and should be accounted for in experimental design.

Limitations
We analyzed protein content of the only HMEF approved for use at our institution. It may be that noncellulose lters or other brands of cellulose material lters could have lesser degrees of protein degradation. We did not directly centrifuge lters as McNeil, et al (5) did as the safety of doing so in a bucket rotor centrifuge was uncertain.