Ryan Walsh (Presenter)
University of Colorado, Denver (AMC)
Authorship: Ryan Walsh and Mark Duncan
(1) University of Colorado, Denver Department of Endocrinology, Metabolism, and Diabetes (2) University of Colorado, Denver Department of Structural Biology and Biochemistry
| Short Abstract Identifying biochemical markers for disease is a difficult endeavor. It requires methods that can quantify multiple components simultaneously, are high throughput, cost effective and deliver high precision and accuracy. We and others are exploring the potential of matrix-assisted laser desorption/ionization (MALDI) for these applications. The approach developed in this study is a non-targeted MALDI based method to analyze human tear fluid. Our results demonstrate that precise measurements are achievable (CV values 10% or less) despite the challenges of internal standard selection, sample handling, and ion suppression. This method gives us the ability to detect, identify, and quantify biomarkers that relate to eye injury and disease. The results of this study will further reinforce the effectiveness of tear as a diagnostic tool and the potential of MALDI-TOF MS for clinical applications. |
Long Abstract
Identifying biochemical markers for disease is a difficult endeavor. It requires a method that can quantify multiple components simultaneously, is high throughput, cost effective and delivers high precision and accuracy. Ideally, the same method should also be applicable in a clinical setting. We and others are exploring the potential of matrix-assisted laser desorption/ionization (MALDI) for these applications. Our results demonstrate that precise measurements are achievable (CV values of 10% or less) despite the challenges of internal standard selection, sample handling, and ion suppression.
We are adopting a non-targeted MALDI profiling approach to quantify the peptides in human tear fluid. Tear can be collected non-invasively (1-20ul) and is rich in potential biomarkers, including peptides and proteins. MALDI-TOF MS is easy to use, analysis times are short and the approach is sensitive, reproducible, and quantitative. There are, however, some significant challenges associated with translating data into accurate quantitative information.
In this study, polystyrene wicks were used to collect tear directly from the surface of the eye. The tear samples were frozen until analysis, then dialyzed to remove salts and other low molecular weight components. Replicates of each samples were analyzed using α-cyano-4-hydroxycinnamic acid as a matrix. To date, more than a thousand peptides and proteins have been identified in tear fluid and some of these could potentially serve as disease markers.
With the careful selection and incorporation of an internal standard it is possible to generate both precise relative and absolute quantitative levels on tear components. In this work, we present an optimized approach to collect, store, and analyze, peptides and proteins, in human tear fluid. The strategy we developed is robust, reproducible, and allows us to work with small volumes (1-5ul) of tear. The results of this study could serve as a diagnostic tool by identifying biochemical markers of eye injury and disease.
References & Acknowledgements:
Acknowledgements
Biodesix
Dr. Senait Asmellash
Kevin Sayers
SimulTOF
Dr. Marvin Vestal
Kevin Hayden
Kenneth Parker
Steven Hatten
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