Phosphotyrosine profiling of human cerebrospinal fluid

Abstract

Background: Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders.

Methods: In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach.

Results: To date, there are no published studies on global profiling of phosphotyrosine modifications of CSF proteins. We carried out phosphotyrosine profiling of CSF using an anti-phosphotyrosine antibody-based enrichment and analysis using high resolution Orbitrap Fusion Lumos mass spectrometer. We identified 111 phosphotyrosine peptides mapping to 66 proteins, which included 24 proteins which have not been identified in CSF previously. We then validated a set of 5 tyrosine phosphorylated peptides in an independent set of CSF samples from cognitively normal subjects, using a PRM-based targeted approach.

Conclusions: The findings from this deep phosphotyrosine profiling of CSF samples have the potential to identify novel disease-related phosphotyrosine-containing peptides in CSF.

Keywords: CSF; Phosphotyrosine; Proteome.

Fig. 1

A schematic of the workflow used to study the phosphotyrosine profiling of CSF. Phosphotyrosine peptides from CSF of three NPH patients were enriched using antibody-based approach and enriched peptides were analyzed on mass spectrometer. The subset of phosphotyrosine sites identified in our discovery experiment were validated in the another set of 8 normal individual CSF using parallel reaction monitoring (PRM) assays

Fig. 2

Summary of phosphotyrosine-containing peptides/proteins in CSF. a Overlap of phosphotyrosine peptides identified from three individuals. We identified 18 phosphopeptides common across the CSF of three individuals, b localization of tyrosine phosphorylated proteins identified in CSF of NPH patients. c Functional categorization of tyrosine phosphorylated protein identified in CSF based on the molecular function, d functional categorization of tyrosine phosphorylated protein identified in CSF based on the biological processes

Fig. 3

MS/MS spectra for the phosphotyrosine-containing peptides/proteins uniquely identified in ours study. a MS/MS spectra for doubly phosphorylated peptide identified from CWF19, b representative MS/MS spectra for phosphorylated peptide identified from G6PD, c representative MS/MS spectra for phosphorylated peptide identified from PTPN6 and d representative MS/MS spectra for phosphorylated peptide identified from PXN

Fig. 4

Validation of tyrosine phosphorylated peptides in control CSF samples by parallel reaction monitoring. a Diazepam binding inhibitor, acyl-CoA binding protein (DBI), b transferrin (TF) and c CD 59 and d beta-1,4-glucuronyltransferase 1(B4GAT1)