Screening Method Using Selected Reaction Monitoring for Targeted Proteomics Studies of Nasal Lavage Fluid

Abstract

Proteomic-based studies of nasal lavage fluid (NLF) may identify molecular pathways associated with disease pathology and new biomarker candidates of upper airway diseases. However, most studies have used rather tedious untargeted MS techniques. Selected reaction monitoring (SRM) is a sensitive and specific technique that can be used with high throughput. In this study, we developed a semiquantitative SRM-based method targeting 244 NLF proteins. The protein set was identified through a literature study in combination with untargeted LC-MS/MS analyses of trypsin-digested NLF samples. The SRM assays were designed using MS/MS data either downloaded from a proteomic data repository or experimentally obtained. Each protein is represented by one to five peptides, resulting in 708 SRM assays. Three to four transitions per assay were used to ensure analyte specificity. The majority (69%) of the assays showed good within-day precision (coefficient of variation ≤ 20%). The accuracy of the method was evaluated by analyzing four samples prepared with varying amounts of four proteins. Peptide and protein ratios were in good agreement with expected ratios. In conclusion, a high throughput screening method for relative quantification of 244 NLF proteins was developed. The method should be of general use in any proteomic study of the upper airways.

A rapid differential display analysis of nasal swab fingerprints to distinguish allergic from non‐allergic rhinitis subjects by mesoporous silica particles and MALDI‐TOF mass spectrometry

“…To the best of our knowledge only few investigations are present in literature about the NF protein composition in the turbinates . The greatest of specific literature is focused on NLF or nasal secretions and nasal epithelium or nasal mucosa . The inferior turbinate plays a pivotal role in the air conditioning activity of the nasal cavity (http://care.american-rhinologic.org/nasal_anatomy; last update: 01/20/2015).…”

Improved preparation of nasal lavage fluid (NLF) as a noninvasive sample for proteomic biomarker discovery

“…As an alternative to theses sample sources, nasal lavage fluids (NLF) and bronchioalveolar lavage fluids (BALF) recently arose showing promising data for a supplementary way for biomarker discovery. Especially for NLF, multiple studies monitored alterations caused by disease states of the airways like asthma [5], sinusitis [6], cystic fibrosis [7], seasonal allergic rhinitis [8][9][10] or general respiratory disorders [11][12][13][14]. All these studies prove the validity of NLF as a means for the identification of biomarkers of diverse disease states.…”

“…Typical candidates of the second group are S100 calcium binding protein A8 and A9, 14-3-3-sigma, triosephosphate-isomerase 1 (TPI1), gamma actin (ACTG1), lipocalin 15 (LCN15), rho GDP dissociation inhibitor beta (ARHGDIB) and fatty acid binding protein 5 (FABP5). To illustrate proteins of the second group, the methods chosen by others vary from tryptic digestion followed by strong cation exchange chromatography and reversed phase chromatography coupled online with MS/MS detection [44], combining of affinity anion exchange, RP-chromatography prior to digestion and LC-MS/MS analysis [8], by labeling techniques like isobaric tags for relative and absolute quantification (iTRAQ) [36] or even selected reaction monitoring [14]. In addition, a gel LC-MS/MS approach was performed to estimate the value and outcome of our modified NLF preparation.…”

Apolipoproteins have a potential role in nasal mucus of allergic rhinitis patients: A proteomic study

“…Nasal mucus proteins in particular have potential as novel biomarkers that could open new therapeutic strategies. Since nasal mucus contains hundreds of different proteins, proteomic techniques could be applied to meet the challenge of identifying key proteins or protein groups …”

“…Little is known about the role of apolipoproteins in allergic rhinitis, although they were discovered in healthy adults' nasal fluid and found to be elevated in allergic rhinitis patients in our previous study . Here, we validated our finding with a another label‐free proteomics approach (based on areas under the curve of extracted ion chromatograms instead of spectral counting) and investigated the source of elevated apolipoproteins in allergic rhinitis by analyzing abundances of selected apolipoproteins in nasal mucus as compared to plasma concentrations in order to determine whether the high abundance in mucus is due to plasma exudation or a local mechanism.…”