RESUMEN
Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer's recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150-200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.
RESUMEN
BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multisystem illness without a currently recognized pharmacological treatment. Dietary supplementation and modification have been posited as potential management strategies; however, their efficacy is controversial. OBJECTIVE: This study aimed to assess the nutritional intake and supplement use of Australian ME/CFS patients and the perceived effect on health-related quality of life (HRQoL) for the first time in an Australian patient population. DESIGN: Between February 2019 and January 2020, ME/CFS patients across Australia volunteered in this cross-sectional study in response to online advertisements. Eligible respondents were invited to complete three online self-administered questionnaires investigating their supplement use, nutritional intake, and HRQoL. The study participants' supplement use and nutritional intake were summarized and compared with the population data returned from the Australian Health Survey (2011-2012). Multiple linear regression analysis was also performed to determine the effect of participants' supplement use and nutrient intake on HRQoL. RESULTS: Twenty-four eligible ME/CFS patients (54.2% meeting the International Consensus Criteria, 79.2% female, mean age = 43.4 ± 10.5 years) completed the online questionnaires. Supplement use was highly prevalent among the study sample (87.5%) and considerably more common when compared with population data (31.9%). Daily total fats and caffeine intakes were significantly higher among ME/CFS patients when compared with the Australian population (P = 0.009 and P = 0.033, respectively), whereas daily intakes of total carbohydrates and alcohol were significantly lower (both P < 0.001). No consistent trends between nutrition and supplement use with patients' HRQoL could be identified. CONCLUSIONS: The daily diet and supplement use of ME/CFS patients appear to vary considerably from those of the general Australian population. Although the role of nutritional intake and supplement use on ME/CFS patients' HRQoL remains unclear, dietary changes and the use of supplements appear to be of value to ME/CFS patients.
