NIIM Kyolic aerobic fitness study

The analysis of human urine samples supplied by the National Institute of Integrative Medicine (NIIMS) for targeted proteomics/polypeptide analysis using a high-resolution LC-MS. The approached described herein is modified from the method by Zimmerli et al. (2008).\nLineage: Proteins (larger than 3KDa) were removed from urine samples by Amicon Centrifugal Filter Units. Urine samples were normalized to the measured creatinine concentration and urine samples were dried in a speed-Vac. Sample pellets were resuspended in 40 uL of 0.1% formic acid in preparation for LC-MS analysis; 5 uL was injected onto the LCMS for analysis.\n\nPolypeptides were analysed in human urine samples following a targeted proteomics (polypeptide) approach using high-resolution LC-MS. The approach described herein is a modified method from Zimmerli et al. (2008), where polypeptides were analysed using an LC-MS Orbitrap Fusion and identified using ProSight 3.0 (Thermo Scientific) for de novo sequencing of peptides and Proteome Discoverer 2.2 for quantification against in-house and UniProtKB libraries. Noting that the point of difference between our approaches and the one performed by Zimmerli et al. is the analytical platform utilised. Zimmerli et al. primarily used Capillary Electrophoresis (CE) coupled to a Time of Flight (ToF) MS for the comparative analysis, but they also used Orbitrap with electron transfer dissociation (ETD) for peptide identification. We are proposing a Liquid Chromatography (LC) coupled to an Orbitrap Fusion MS to enable simultaneous untargeted identification and comparative semi-quantitative analysis. \n\nSamples were separated in an Advance Bio Peptide Map column (2.1 x 150 mm, 2.7 µm, Agilent Technologies) using Vanquish UHPLC system (Thermo Scientific) and analysed by Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Urinary peptides were identified by Proteome Discoverer 2.2 (Thermo Scientific). A list of 81 urinary peptides identified by Brown et al. (2015) was used to search peptides in the urine samples.