Identification of novel biomarker candidates by proteomic analysis of cerebrospinal fluid from patients with moyamoya disease using SELDI-TOF-MS

Abstract

Background: Moyamoya disease (MMD) is an uncommon cerebrovascular condition with unknown etiology characterized by slowly progressive stenosis or occlusion of the bilateral internal carotid arteries associated with an abnormal vascular network. MMD is a major cause of stroke, specifically in the younger population. Diagnosis is based on only radiological features as no other clinical data are available. The purpose of this study was to identify novel biomarker candidate proteins differentially expressed in the cerebrospinal fluid (CSF) of patients with MMD using proteomic analysis.

Methods: For detection of biomarkers, CSF samples were obtained from 20 patients with MMD and 12 control patients. Mass spectral data were generated by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) with an anion exchange chip in three different buffer conditions. After expression difference mapping was undertaken using the obtained protein profiles, a comparative analysis was performed.

Results: A statistically significant number of proteins (34) were recognized as single biomarker candidate proteins which were differentially detected in the CSF of patients with MMD, compared to the control patients (p < 0.05). All peak intensity profiles of the biomarker candidates underwent classification and regression tree (CART) analysis to produce prediction models. Two important biomarkers could successfully classify the patients with MMD and control patients.

Conclusions: In this study, several novel biomarker candidate proteins differentially expressed in the CSF of patients with MMD were identified by a recently developed proteomic approach. This is a pilot study of CSF proteomics for MMD using SELDI technology. These biomarker candidates have the potential to shed light on the underlying pathogenesis of MMD.

Figure 1

Mass spectra of representative single biomarker candidate proteins in CSF under different pH conditions. Protein profiles of the MMD and control groups were generated using Q10 (strong anion exchanger) array. For each pH condition, the upper two spectra are protein profiles obtained between m/z 2,000 and 10,000, and the lower two spectra are expansions showing the peak intensities around m/z 4473, 4588 and 4476 for pH 5, 7 and 9, respectively. All representative peaks (red arrows) are larger for the MMD than control group under each pH condition, as determined by SELDI-TOF-MS.

Figure 2

Peak intensity of representative single biomarker candidate proteins in the MMD and control groups. Peak intensities of representative single biomarker candidate proteins detected using SELDI-TOF-MS are significantly larger in the MMD than control group under each pH condition (m/z 4473, 4588 and 4476 for pH 5, 7 and 9, respectively). The box-whisker plots indicate the median value (thick line) and the 25th (lower line of box) and 75th (upper line of box) percentile; T bars indicate the 10th and 90th percentile. The p values between the groups were calculated using Mann-Whitney U test. *P < 0.01.

Figure 3

CART analysis using peaks obtained by SELDI-TOF-MS to discriminate between patients with MMD and control patients. The decision tree was constructed using CSF samples from 32 patients with MMD and control patients. The classification is determined starting at the roof node, following by appropriate splitting decisions based on the peak intensity at each node. If the peak intensity is lower than the cutoff intensity value, the left node is selected. This splitting process is continued until no further classification is achieved and terminal nodes are produced. Using m/z 4473, 2406 and 6338 peaks (pH 5), m/z 4588 and 7250 peaks (pH 7), and m/z 4746 and 1044 peaks (pH 9), CART for Q10 ProteinChip was applied to identify patients with MMD and control patients. The analysis correctly classified all 20 patients with MMD under pH 5 condition and 19 of 20 under the pH 7 and 9 conditions; all 12 control patients were classified under all pH conditions.

Figure 4

CART analyses using all 34 single biomarker candidate proteins. CART was analyzed for 34 single biomarker candidate proteins identified in the CSF under each pH condition (pH 5, 7 and 9) to discriminate patients with MMD from control patients. This analysis correctly classified 19 of 20 patients with MMD and all 12 control patients based on the peak intensities of the m/z 4473 peak (pH 5) and m/z 4588 peak (pH 7).