Proteomic analysis of single tears from healthy human subjects

ABSTRACT: 23 volunteers were recruited for this study. Tears were collected using microcapillary tubes (MCT). For each volunteer, a single tear sample was taken from one eye. For two of these volunteers, the procedure was performed in the morning (9-10 AM) and in the afternoon (5-6 PM) on the same day, once a week for 3 consecutive weeks. Proteins were purified from 5 uL of tear sample using the methanol-chloroform precipitation protocol. The lyophilized proteins were resuspended in ammonium bicarbonate 50 mM, pH 8. An in-solution trypsin digestion was performed. For each run, 1 ug of peptides was injected and each sample was injected twice. Analyses were performed by LC-MS/MS using an Orbitrap Fusion mass spectrometer coupled to an EASY-nLC 1000 nanoflow high-pressure liquid chromatography. Peptides were separated on a commercial analytical nanocolumn. MS scans were performed using the Orbitrap (OT) analyzer with a resolution of 120 000. Precursor ions were selected for higher collisional dissociation (HCD) and the collision energy was set to 30%. Dynamic exclusion was set at 45 s, with a repeat count of 1.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Erika Ponzini  

PROVIDER: MSV000087703 | MassIVE | Fri Jun 25 08:03:00 BST 2021

REPOSITORIES: MassIVE