Overview of proteomics studies in obstructive sleep apnea

Abstract

Obstructive sleep apnea (OSA) is an underdiagnosed common public health concern causing deleterious effects on metabolic and cardiovascular health. Although much has been learned regarding the pathophysiology and consequences of OSA in the past decades, the molecular mechanisms associated with such processes remain poorly defined. The advanced high-throughput proteomics-based technologies have become a fundamental approach for identifying novel disease mediators as potential diagnostic and therapeutic targets for many diseases, including OSA. Here, we briefly review OSA pathophysiology and the technological advances in proteomics and the first results of its application to address critical issues in the OSA field.

Keywords: Biomarkers; Lung; Mass spectrometry; Metabolic disorders; Obstructive sleep apnea; Proteomics.

Fig. 1

Discovery-based strategy for candidate biomarker of Obstructive Sleep Apnea. In discovery-based proteomics approach, the 2D-difference gel electrophoresis (2-DIGE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (Shotgun) are the most popular methodologies to identify disease candidate biomarkers. The proteins identified as differentially abundant (up/down and present/absent) in disease are verified by orthogonal methodologies (other than those used in the discovery phase) before proceeding to the next validation phase in a larger cohort of patients and translation into clinical application.

Fig. 2

Simplified scheme of a typical mass spectrometry (MS)-based strategy to identify potential biomarkers (bottom up proteomics). In the first step proteins (A) are digested (B) to peptides (C) usually by trypsin (which predominantly cleaves at the carboxyl side of lysine “K” and arginine “R” except when either is followed by proline). (D) The tryptic peptides generated from protein digestion are injected into a liquid chromatography system (for peptide separation) online coupled with a mass spectrometer, which employs two stages of mass analysis (tandem MS, MS/MS). (E) The first stage is MS which corresponds to mass scans of peptides eluted at different time points from the LC system. (F) In the second stage, some peptides, usually the most abundant, are automatically selected for fragmentation generating MS/MS spectra. Mass differences between peaks in the MS/MS spectrum can be directly correlated to amino acid residues and a short peptide sequence can be obtained. Protein and peptide identification is achieved by computer-based matching of the experimental tandem mass spectra with the theoretical tandem mass spectra generated from in silico digestion of protein sequence databases.

Fig. 3

Annotated raw tandem mass spectrometry (MS/MS) spectrum (real example of Fig. 2F) of the peptide “TyFPHFDLSHGSAQVK nitrosylation” from hemoglobin subunit alpha with tyrosine nitrosylated. Protein extracts of OSA erythrocyte cells were digested by trypsin followed by liquid chromatography (LC)–MS/MS analysis. The peptide “TyFPHFDLSHGSAQVK nitrosylation” was observed at charge state z ≥ +3 in scan number 24071 (which corresponded to a specific elution time from the LC system) and at 621.6344 m/z. The x-axis is mass over charge (m/z) for fragment ions and the y-axis is intensity counts. Theoretical fragment ions that match the observed fragments in the MS/MS spectrum are annotated with blue text. Amino acid residues can be correlated to mass difference between peaks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)