Title: Liquid chromatography tandem mass spectrometry (LC-MS/MS) of cerebrospinal fluid identifies proteins and pathways altered in ALS Additional authors (in order): Collins MA, An J
Institution name, city, state, country of additional authors: Barrow Neurological Institute, Phoenix, AZ
Background: There continues to be a need for ALS biomarkers to uncover mechanisms of disease and for use in clinical trials to monitor treatment effects or stratify the patient population to identify those that may best respond to treatment. Cerebrospinal fluid (CSF) is a rich source for biomarkers as a biofluid that is proximal to the site of disease and a matrix much less complex than blood. Hypothesis: We propose that an unbiased proteomic analysis of CSF from ALS, healthy and disease controls will identify proteins and pathways specifically altered in ALS patients. Methods: CSF was obtained from the NEALS Biorepository from a total of 90 sporadic ALS patients, 20 familial ALS patients, 80 healthy controls, and 60 disease controls (20 AD, 20 MS, 10 upper motor neuron disease, 10 lower motor neuron disease). Since LC-MS/MS is not a high throughput technique, we generated pooled CSF samples within each subject group, each pool consisting of 10 age- and gendermatched subjects. Therefore, our experimental samples consisted of 9 pools from sALS, 2 pools from fALS, 8 from healthy controls, 2 AD, 2 MS, 1 upper motor neuron disease and 1 lower motor neuron disease. After removing the 6 most abundant proteins from the CSF pools, LC-MS/MS was performed in triplicate on all pooled samples. A separate set of 9 individual sALS, 7 fALS, and 4 healthy controls were used as a validation set. Results: A total of 1,712 proteins were reliably identified that fit specific inclusion criteria (required number of unique peptides detected in a minimum number of samples). Several proteins with diverse biological functions were significantly altered in ALS. Pathway analysis identified inflammation, neuronal activity, and extracellular matrix regulation as key biologic pathways altered in ALS. Our findings were validated in a separate sample set by LC-MS/MS and select proteins further validated by western blot and immunohistochemistry of spinal cord tissue sections from ALS and control subjects. Conclusions: Our results illustrate the utility of CSF proteomic profiling for identifying ALS protein and pathways altered in disease and new candidate biomarkers for ALS, including tenascin R, SPARCL1, and eIF 4e.
