A Parallel Reaction Monitoring Mass Spectrometric Method for Analysis of Potential CSF Biomarkers for Alzheimer's Disease

Abstract

Scope: The aim of this study was to develop and evaluate a parallel reaction monitoring mass spectrometry (PRM-MS) assay consisting of a panel of potential protein biomarkers in cerebrospinal fluid (CSF). Experimental design: Thirteen proteins were selected based on their association with neurodegenerative diseases and involvement in synaptic function, secretory vesicle function, or innate immune system. CSF samples were digested and two to three peptides per protein were quantified using stable isotope-labeled peptide standards. Results: Coefficients of variation were generally below 15%. Clinical evaluation was performed on a cohort of 10 patients with Alzheimer's disease (AD) and 15 healthy subjects. Investigated proteins of the granin family exhibited the largest difference between the patient groups. Secretogranin-2 (p<0.005) and neurosecretory protein VGF (p<0.001) concentrations were lowered in AD. For chromogranin A, two of three peptides had significantly lowered AD concentrations (p<0.01). The concentrations of the synaptic proteins neurexin-1 and neuronal pentraxin-1, as well as neurofascin were also significantly lowered in AD (p<0.05). The other investigated proteins, β2-microglobulin, cystatin C, amyloid precursor protein, lysozyme C, neurexin-2, neurexin-3, and neurocan core protein, were not significantly altered. Conclusion and clinical relevance: PRM-MS of protein panels is a valuable tool to evaluate biomarker candidates for neurodegenerative disorders.

Synaptic proteins in CSF as potential novel biomarkers for prognosis in prodromal Alzheimer’s disease

“…Several of these proteins, including VGF, CHGA, SCG2, CysC, and β 2 M, have been suggested in previous studies to be involved in AD pathology [12][13][14][22][23][24][25][26][27][28]. A pilot study that we performed after developing the PRM panel showed promising results, with lower levels for several proteins in patients with AD dementia compared with control subjects [29]. The aims of the present study were to validate this panel of proteins in a larger and independent cohort, as well as to investigate whether the panel has potential for distinguishing patients with prodromal AD and patients with AD dementia from cognitively healthy subjects.…”

“…The sample preparation and acquisition methods for PRM analysis are described in detail elsewhere [29]. For the present study, we used the same sample preparation and MS methods, with minor modifications as described below.…”

“…To adjust for these effects, a CSF pool was aliquoted, and four pool samples were used as calibrants in each run of 40 samples. Study samples, CSF pool samples, and heavy isotopelabeled standards were prepared as previously described [29], except that standard 0.5-, 1.5-, and 2.0-ml polypropylene tubes were used in the present work. After thawing, 20 μl of the internal standard mixture was added to each sample.…”

“…Levels correlated highly with each other between the two PRM panels (Pearson's r = 0.92) and did not differ between diagnostic groups, but they were slightly different between the subsets subjected to trypsinization and PRM analysis on separate occasions (see Additional file 1). CVs of the proteins of the PRM panel are described in detail elsewhere [29]. The CSF pool samples were used to normalize data between acquisition days.…”

“…After thawing, 20 μl of the internal standard mixture was added to each sample. Tryptic digestion was performed as previously described [29], except that no shaking of samples was performed. Digested samples were centrifuged, desalted using Oasis 30-μm HLB 96-well μElution plates (Waters, Milford, MA, USA) with MeOH for elution into polypropylene plates.…”