Two different isolation methods have been used: Background: Methods: To date there is no blood-based diagnostic test for neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and disease recognition relies on the exclusion of other diseases and observation of symptom progression in accordance with a clinician’s experience, implicating a significant delay in diagnosis; A Pooled Plasma sample (PP) has been created using plasma from six Healthy Controls between 51.2-62.9 years of age and with known blood level of NF heavy chain (between 7.0 and 42.9ng/ml). Samples were obtained from control individuals taking part in the ALS biomarkers study (ethical approval: 09/H0703/27); Two different isolation methods have been used: Hallmark of neurodegeneration is the presence of protein aggregates within neurons (1). Impaired blood brain barrier (BBB) would enable the leakage of molecules and macromolecules linked to neurodegeneration into blood (2); • SeprionTM PAD-beads (Microsens Biotechnologies, UK) • Ultracentrifugation using Sorvall Discovery 100SE equipped with a TFT-80.2 rotor. Plasma has been incubated with Triton X-100 and added on top of a solution 1M sucrose and spin at 50000rpm for 2 hours at +4°C. Pellet has been collected; Neurofilaments have been proved to be good biomarkers of disease progression in ALS and in other neurodegenerative disorders. Samples produced with ultracentrifugation and SeprionTM PAD-beads have been analysed by Western Blot, LC-MS/MS (Orbitrap Velos Pro) and Immunogold-TEM. Data from optimization of NF immune-assays in ALS suggest that NF may be included in circulating protein aggregates which may interfere with their detection. Circulating protein aggregates may be informative of a disease state; hence their isolation and characterization may be crucial for the development of a novel breed of disease biomarkers. References: 1.Blokhuis AM, Groen EJ, Koppers M, van den Berg LH, Pasterkamp RJ. Protein aggregation in amyotrophic lateral sclerosis. Acta neuropathologica. 2013;125(6):777-94. Objective: 2.Garbuzova-Davis S, Hernandez-Ontiveros DG, Rodrigues MC, Haller E, Frisina-Deyo A, Mirtyl S, et al. Impaired blood-brain/spinal cord barrier in ALS patients. Brain research. 2012;1469:114-28. Develop a method for isolation of circulating protein aggregates from plasma Anti-NFH Anti-NFM Anti-NFL Amikon Filter 100K Ultracentrifugation SeprionTM (a) (b) (c) 460 268 238 171 117 71 55 41 31 Figure1. Western Blots showing presence of NFs within PP sample after protein concentration through Amicon Filter 100K (Millipore) (a), after aggregates isolation through ultracentrifugation (b) and SeprionTM PAD-beads (c). Figure2. (a) SDS-PAGE shows the fractions generated for in-gel Trypsin digestion, followed by LC-MS/MS. (b) The following LC-MS/MS generated 650 and 1068 proteins for UC_PP and SEP_PP, respectively (Protein Grouping: True; Peptide Confidence: High; Minimal number of peptides: 1). No Neurofilament proteins have been detected by LC-MS/MS. UC_PP1 UC_PP2 UC_PP3 UC_PP4 UC_PP5 UC_PP6 UC_PP7 UC_PP8 UC_PP9 UC_PP10 UC_PP11 UC_PP12 UC_PP13 UC_PP14 UC_PP15 SEP_PP1 SEP_PP2 SEP_PP3 SEP_PP4 SEP_PP5 SEP_PP6 SEP_PP7 SEP_PP8 SEP_PP9 SEP_PP10 SEP_PP11 SEP_PP12 SEP_PP13 SEP_PP14 SEP_PP15 460 268 238 171 117 71 55 41 31 (a) (b) Figure3. The proteins found by LC-MS/MS have been classified using PANTHER (Protein ANalysis THrough Evolutionary Relationships, http://pantherdb.org/). From the shared fraction protein list is clear that these two methods isolate cytoskeletal and other intracellular proteins, potential allowing the detection of CNS derived material in blood. In addition, this fraction presents chaperones and nucleic acid binding proteins, which are known play a role in ALS. Shared fraction Exclusive UC fraction Exclusive SEP fraction Anti-NFH Anti-NFM Anti-NFL Figure4. The images displayed were produced by Immunogold Electron Microscopy to validate the presence of aggregates containing NFs and they seem to replicate data produced by Western Blot. In particular, the three proteins are all present in UC_PP with NFH mainly present within big aggregates. UC_PP SEP_PP Conclusion Acknowledgements The two methods are suitable for isolation of protein aggregates containing cytoskeletal and other cytosolic proteins from blood plasma This study is funded by a MRC Industry CASE Studentship, shared between Queen Mary University of London and Proteome Sciences. Ultracentrifugation is the best method for isolation of protein aggregates containing NFs due to its specificity and clear pellets produced Plasma samples obtained from study 09/H0703/27. Ultracentrifugation will be performed as standard method to isolate protein aggregates from ALS patients' blood plasma. The fractions generated will be studied in order to generate a panel of candidates for diagnosis of ALS.