Development of Parallel Reaction Monitoring (PRM) Assays for the Validation of Biomarkers Associated to Alzheimer’s Disease in CSF Claudia Fredolini (Presenter) KTH – Royal Institute of Technology >> POSTER (PDF) Presenter Bio: In 2008 I started my PhD studies in Molecular Medicine at the University of Turin (Italy) and I joined the Italy-USA Oncoproteomics Program, collaboration between the Italian Institute of Health and the Center for Applied Proteomic and Molecular Medicine (CAPMM) at the George Mason University. At CAPMM I was engaged for four years in research on cutting-edge technologies for diagnostics and personalized medicine. There I contributed to the development of a new technology (Nanotrap®) for high sensitivity measurement of proteins in biological fluids. After completing the PhD in 2012, I kept working at the CAPMM for about one year as Research Assistant Professor and then I decided to move back in Europe joining the Scilifelab in Stockholm where I worked in an interdisciplinary project aimed to integrate mass spectrometry and antibody based assays to enhance the translation of biomarkers discovery into verification and validation. Over the past ten years I developed a strong interest in clinical chemistry and translational medicine and I had the chance of experiencing a wide range of technologies, exploring and testing their potential and limits as analytical methods in research and eventually as diagnostic tools. I am very interested in expanding my knowledge and research into novel molecular techniques for the detection and quantification of biomolecules in clinical samples, and I recently joined the Molecular Tool program at the University of Uppsala (Department of Immunology, Genetics and Pathology). Besides being active as researcher (28 publications and 3 submitted or in preparation), during the past ten years of academic experience I also had the opportunity to serve as teaching assistant, guest lecturer, mentor and supervisor for undergraduate and PhD level students, in Italy, USA and Sweden.

Authors: Annika Andersen (1),Julia Remnestål (1) ,Fredrik Edfors (1), Mathias Uhlén (1), Jochen Schwenk (1), Anna Häggmark-Månberg (1), Peter Nilsson (1) and Claudia Fredolini (1)(2)
(1) SciLifeLab, KTH – Royal Institute of Technology, Stockholm, Sweden , (2) Department of Immunology, Genetics and Pathology and SciLifeLab, Uppsala University

Suspension bead arrays (SBA) have shown to be a convenient and successful method for protein profiling of cerebrospinal fluid (CSF) in the contest of Alzheimer’s disease. Nevertheless, robust and specific orthogonal analytical methods are needed to support the verification of antibody based discoveries. We developed and validate parallel reaction monitoring (PRM) assays for 17 brain enriched proteins previously profiled by SBA in a cohort of 92 CSF samples from Alzheimer’s disease (AD) patients and control individuals. In order to verify the differential profiles observed by affinity proteomics, PRM assays were used to quantify the proteins in the same cohort.

Introduction

Suspension bead array (SBA) has shown to be a convenient and successful method for protein profiling of cerebrospinal fluid (CSF), supporting the discovery of novel candidate biomarkers for Alzheimer’s disease. Nevertheless high-throughput and high level of multiplexing in single binder assays are gained at expenses of target specificity due to occasional antibody cross-reactivity and/or off-target binding. Robust and specific alternative assays are needed to support a rapid verification of antibody based protein profiles and eventually the translation of the most robust candidate biomarkers into a validation phase.

Here we developed and validated parallel reaction monitoring (PRM) analytical assays for the quantification of 17 CSF brain enriched proteins previously profiled in Alzheimer’s disease (AD) patients and control individuals by SBA analysis [1]. SBA data were then compared with PRM measurement obtained in the same cohort of samples.

Methods

PRM target mass spectrometry (MS) assays were developed and validated using CSF from healthy donors and pools of CSF samples from AD patients. Samples were spiked-in with heavy labeled protein fragments (QprESTs) [2], reduced, alkylated and then digested using a mixture of Trypsin and LysC. Assay development included evaluation of different aspects in the sample preparation such as digestion time and peptides clean-up procedure. In the final protocol, semi-automated enzymatic digestion and purification by µElution HLB (Hydrophilic-lipophilic balance) SPE plates were implemented for high throughput sample preparation. MS analysis was performed using a Q-Exactive HF (Thermo) equipped with an Ultimate 3000 RSLC nanosystem (Dionex). Samples were injected into a C18 guard desalting column and then into a 25 cm x 75 µm ID, particle size 2 µm, Thermo Easy spray analytical column (Thermo).PRM parameters were optimized for 34 peptides. Raw data were imported into Skyline for peak detection. Data analysis and representation was performed on the environment for statistical computing and graphics R. Validated method were applied to measure peptide levels in a cohort of 92 cerebrospinal fluid (CSF) samples including 43 AD, 14 Preclinical, 2 Prodromal. Control samples included 14 individuals diagnosed with non-AD mild cognitive impairment (MCI) and 23 cognitively normal.

Results

PRM assays for 17 brain enriched proteins detectable in CSF were developed nevertheless a thorough method validation and evaluation was performed only for 11 targets, those higher abundant. Validated methods show good linearity (average R2 = 0.999-0.970), high intra-day (coefficient of variance (CV) = 3%-11%), and medium high inter-day precision (coefficient of variance (CV) = 20%-30%). Protein measurement was performed by PRM for all the 17 proteins (34 peptides) in the cohort of 92 CSF samples including AD patients and control individuals. Antibody profiling data and PRM measurement strongly correlated for 6 out 17 proteins (Pearson’s coefficient > 0.70) and moderately for 2 proteins (0.50< Pearson’s <0.70) . When not correlating with SBA data, PRM protein quantification revealed anyway clinically interesting profiles, complementing the information obtained by SBA analysis.

Conclusions & Discussion

PRM target MS assays, featuring exquisite specificity and multiplex capacity, support successfully the verification of SBA discoveries for medium high abundant proteins in un-depleted CSF. PRM target MS assays moreover, complement antibody based discoveries elucidating novel clinically interesting profiles. The PRM analytical methods developed in a semi-automated fashion are suitable for larger validation studies.

[1] Remnestål J, Just D, Mitsios N, Fredolini C, Mulder J, Schwenk JM, et al. CSF profiling of the human brain enriched proteome reveals associations of neuromodulin and neurogranin to Alzheimer's disease. Proteomics Clin Appl 2016;10:1242-1253.

[2] Tove Boström Nature Methods 13 (2016)