on 06-19-201704:04 AM - edited on 10-15-202106:10 AM by Closed Account
Daniel Lopez-Ferrer1, Romain Huguet1, Ioanna Ntai1, David Horn1, Peter Raus2, Vlad Zabrouskov1, Andreas FR Huhmer1 and Peter Verhaert3 ASMS 2017 Poster Purpose: Demonstrate the potential of top-down proteomics for mapping the proteoform diversity in the tear fluid. The proposed research workflow includes the use of Schirmer Tear Test strips (Clement Clarke International, Harlow UK) for sample collection, sample handling, and protein extraction in conjunction with various top-down fragmentation techniques on a chromatographic time scale.
Methods: Tear proteins extracted from the Schirmer strips in an aqueous solution are directly analyzed by LC-MS using an Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer modified with UVPD. MS/MS acquisition was performed using ETD, EThcD, HCD and UVPD fragmentations, detecting precursor and fragment ions with a resolution of 120,000 at m/z 200. Data analysis was performed using Thermo Scientific™ Deconvolution™ 4.0 software and ProsightPD nodes in the Thermo Scientific™ Proteome Discoverer 2.2 software.
Results: Our approach consists of a simplified two-step workflow. First, proteins are extracted from the Schirmer strips using a water-based buffer. Samples are then analyzed over a 30 or 60 min LC gradient by MS and MS/MS using various fragmentation mechanisms. One of the proteins represented in the dataset by a multitude of proteoforms is lacritin, which is a prosecretory mitogen capable of promoting basal tearing and low levels of which are associated with dry eye syndrome. Overall, this strategy offers a powerful option for discovery and characterization of potential tear biomarkers that could be used for the screening of diseases, both eye related or other.
1 Thermo Fisher Scientific, San Jose, California, USA
2 MirÃ³ Center for Oculoplastic Surgery, Geel, Belgium
3 ProteoFormiX, Beerse, Belgium.