Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay

Background

Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer’s Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer’s and Parkinson’s disease (PD).

Results

Thirty eight proteins showed significantly (p?<?0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p?<?0.0001), lysosome-associated membrane protein 1 (p?<?0.0001), pro-orexin (p?<?0.0017) and transthyretin (p?<?0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM_2?activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r²?≥?0.39, p?≤?0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r²?≥?0.4, p?≤?0.03) with phosphorylated–tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups.

Conclusions

Using proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.

     

An assessment of sample processing methods for stable isotope analyses of marine food webs

Carbon and nitrogen stable isotope ratios are commonly used in the study of marine food webs. However, different sample processing methods can influence the measurement of these stable isotope ratios. The purpose of this study is to define an adequate methodology to be used in the construction of whole food webs. It is demonstrated that acidification of the samples results in a decrease in carbon stable isotope values for sedimentary organic matter, suspended particulate organic matter, plankton and invertebrates with carbonated structures. The response was variable for nitrogen isotope ratios. Based on our results we recommend sample acidification for carbon analysis in these compartments where effects of this treatment were observed. We observed a decrease in δ¹³C values after washing with distilled water, so we do not recommend washing with water after acidification. For nitrogen analysis, acidification should be avoided. The various dehydration treatments studied caused significant differences only in nitrogen isotope ratios.     

Improving Fab’ fragment retention in an autonucleolytic <Emphasis Type="Italic">Escherichia coli</Emphasis> strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA

Objectives

To reduce unwanted Fab’ leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab’ fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives.

Results

We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab’ grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab’ coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab’ leakage relative to the original autonucleolytic Fab’ strain with OmpA-fused staphylococcal nuclease.

Conclusions

We successfully rescued Fab’ leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab’ fragment to the surrounding growth media.