A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Published: October 20th, 2016

DOI:

Wendy E. Heywood^1*, Anna Baud^1*, Emily Bliss¹, Ernestas Sirka¹, Jonathan M. Schott², Henrik Zetterberg³, Daniela Galimberti⁴, Neil J. Sebire⁵, Kevin Mills¹

¹Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, ²Dementia Research Centre, Institute of Neurology, University College London, ³Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, ⁴Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, ⁵Great Ormond Street Hospital for Children, University College London

* These authors contributed equally

Many neurodegenerative diseases are still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often potential new biomarkers do not make it into the clinic due to limitations in their development and high costs. However, targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).

In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.

The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.