1. 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 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):
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:
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, 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.