An Integrated Workflow for Multiplex CSF Proteomics and PeptidomicsIdentification of Candidate Cerebrospinal Fluid Biomarkers of Alzheimer’s Disease

Many
disease processes in the brain are reflected in the protein
composition of the cerebrospinal fluid (CSF). In addition to proteins,
CSF also contains a large number of endogenous peptides whose potential
as disease biomarkers largely remains to be explored. We have developed
a novel workflow in which multiplex isobaric labeling is used for
simultaneous quantification of endogenous CSF peptides and proteins
by liquid chromatography coupled with mass spectrometry. After the
labeling of CSF samples, endogenous peptides are separated from proteins
by ultrafiltration. The proteins retained on the filters are trypsinized,
and the tryptic peptides are collected separately. We evaluated this
technique in a comparative pilot study of CSF peptide and protein
profiles in eight patients with Alzheimer’s disease (AD) and
eight nondemented controls. We identified several differences between
the AD and control group among endogenous peptides derived from proteins
known to be associated with AD, including neurosecretory protein VGF
(ratios AD/controls 0.45–0.81), integral membrane protein 2B
(ratios AD/controls 0.72–0.84), and metallothionein-3 (ratios
AD/controls 0.51–0.61). Analysis of tryptic peptides identified
several proteins that were altered in the AD group, some of which
have previously been reported as changed in AD, for example, VGF (ratio
AD/controls 0.70).