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.

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Hairdressers are frequently exposed to bleaching powder containing persulfates, a group of compounds that may induce hypersensitivity in the airways. The mechanism causing this reaction is not clear. The aim of this study was to identify changes in the nasal lavage fluid proteome after challenge with potassium persulfate in hairdressers with bleaching powder-associated rhinitis. Furthermore, we aimed to compare their response to that of hairdressers without nasal symptoms, and atopic subjects with pollen-associated nasal symptoms. To study the pathogenesis of persulfate-associated rhinitis, the response in protein expression from the upper airway was assessed by time-dependent proteomic expression analysis of nasal lavage fluids. Samples were prepared by pooling nasal lavage fluids from the groups at different time points after challenge. Samples were depleted of high-abundant proteins, labeled with iTRAQ and analyzed by online 2D-nanoLC-MS/MS. Differences in the protein pattern between the three groups were observed. Most proteins with differentially expressed levels were involved in pathways of lipid transportation and antimicrobial activities. The major finding was increased abundance of apolipoprotein A-1, 20 min postchallenge, detected solely in the group of symptomatic hairdressers. Our results suggest there may be differences between the mechanisms responsible for the rhinitis in the symptomatic and atopic group.

Although obesity and high levels of low-density lipoprotein (LDL) are well-known risk factors for cardiovascular disease, the precise role(s) of different LDL constituents in obesity has not been explored. In the present study, we compared the LDL proteome of healthy control adults (body mass index<25) and obese subjects (body mass index>30). LDL was isolated by density-gradient ultracentrifugation and proteins were separated with 2-D PAGE, quantified, and identified by peptide mass fingerprinting using MALDI-TOF MS. A new LDL-associated protein was identified as transthyretin and found to be significantly more abundant in LDL from the obese subjects. In addition, LDL from the obese subjects contained relatively more α(1) -antitrypsin, apo J, apo C-II, than LDL from controls, and also more of an acidic isoform (pI/Mr; 5.2/23 100) of apo A-I. On the other hand, the relative amounts of apo A-IV and the major isoform of apo A-I (pI/Mr; 5.3/23 100) were significantly less in LDL from the obese subjects. Apo E was less and non-sialylated apo C-III more abundant in LDL from obese men than control men, while there were no such differences between LDL from obese and control women. These findings illustrate that obesity is not only associated with increased LDL-cholesterol levels but also with alterations in the LDL protein composition. The presence of transthyretin in LDL from obese subjects may reflect over-nutrition and affect the lipid metabolism in obesity.

Hairdressers have an increased risk for developing airway symptoms, for example, asthma and rhinitis. Persulfates, which are oxidizing agents in bleaching powder, are considered important causal agents for these symptoms. However, the underlying mechanisms are unclear. The aim was therefore to measure proteomic changes in nasal lavage fluid from persulfate-challenged subjects to identify proteins potentially involved in the pathogenesis of bleaching powder-associated rhinitis or candidate effect biomarkers for persulfate. Also, oxidized peptides were measured to evaluate their usefulness as biomarkers for persulfate exposure or effect, for example, oxidative stress. Samples from hairdressers with and without bleaching powder-associated rhinitis were analyzed with liquid chromatography tandem mass spectrometry using selected reaction monitoring to target 246 proteins and five oxidized peptides. Pathway analysis was applied to obtain a functional overview of the proteins. Several proteins involved in biologically meaningful pathways, functions, or disorders, for example, inflammatory responses, oxidative stress, epithelium integrity, and dermatological disorders, changed after the persulfate challenge. A list with nine proteins that appeared to be affected by the persulfate challenge and should be followed up was defined. An albumin peptide containing oxidized tryptophan increased 2 h and 5 h after the challenge but not after 20 min, which indicates that such peptides may be useful as oxidative stress biomarkers.

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Oxidative stress has been suggested as an underlying mechanism of many human diseases. However, definitive evidence for this association has not been presented due to different shortcomings of the methods used to measure biomarkers of oxidative stress. Persulfates are oxidizing agents known to elicit hypersensitive reactions from the airways and skin. Despite a frequent use of persulfates at many work places, no biomarkers for persulfate exposure are available. The aim of this study was to develop a strategy for the identification and detection of multiple oxidative modifications within proteins. This strategy was applied on persulfate-oxidized proteins to identify oxidized peptides suitable for further investigation as biomarkers of persulfate exposure or oxidative stress. A strategy for the identification and the relative quantification of multiple oxidative modifications within proteins was developed. The usage of two software packages facilitated the search for modified peptides to a great extent. Oxidized peptides were relatively quantified using liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode. The result showed that persulfates oxidize tryptophans and methionines resulting in mass shifts of 16 and/or 32 Da. Also, oxidized albumin peptides in nasal lavage fluid samples from subjects challenged with persulfate were detected. The oxidation degree before and after challenge remained constant for peptides containing methionine sulfoxide. For peptides containing oxidized tryptophan the oxidation degree increased after exposure. Some of these oxidized peptides may be suitable as biomarkers; however, further evaluation is required.

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