Quantitative proteomic analysis of cerebrospinal fluid of women newly diagnosed with multiple sclerosis.

PURPOSE: The lack of reliable diagnostic and/or prognostic biomarkers for multiple sclerosis (MS) is the major obstacle to timely and accurate patient diagnosis in MS patients. To identify new proteins associated with MS we performed a detailed proteomic analysis of cerebrospinal fluid (CSF) of patients newly diagnosed with relapsing-remitting MS (RRMS) and healthy controls. MATERIAL: Reflecting significantly higher prevalence of MS in women we included only women patients and controls in the study. To eliminate a potential effect of therapy on the CSF composition, only the therapy-naïve patients were included. METHODS: Pooled CSF samples were processed in a technical duplicate, and labeled with stable-isotope coded TMT tags. To maximize the proteome coverage, peptide fractionation using 2D-LC preceded mass analysis using Orbitrap Fusion Tribrid Mass Spectrometer. Differential concentration of selected identified proteins between patients and controls was verified using specific antibodies. RESULTS: Of the identified 900 CSF proteins, we found 69 proteins to be differentially abundant between patients and controls. In addition to several proteins identified as differentially abundant in MS patients previously, we observed several linked to MS for the first time, namely eosinophil-derived neurotoxin and Nogo receptor. CONCLUSIONS: Our data confirm differential abundance of several previously proposed protein markers, and provide indirect support for involvement of copper-iron disbalance in MS. Most importantly, we identified two new differentially abundant CSF proteins that seem to be directly connected with myelin loss and axonal damage via TLR2 signaling and Nogo-receptor pathway in women newly diagnosed with RRMS.

Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage.

Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC-MS/MS analysis. LC-MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the "waste" fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded "waste" fractions contain proteins of potential interest and should be included in CSF biomarker studies.