Proteomic analysis of mucopolysaccharidosis I mouse brain with two-dimensional polyacrylamide gel electrophoresis
Introduction
Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease that results from deficiency of α-l-iduronidase (IDUA, E.C.3.2.1.76), which degrades the glycosaminoglycans (GAG) heparan sulfate and dermatan sulfate. The widespread accumulation of GAG leads to progressive cellular damage and organ dysfunction, with the central nervous system (CNS) being one of the primary sites of pathology. The CNS pathology in MPS I patients is manifested by learning delays, dementia, hydrocephalus and mental retardation. The etiology of neurological dysfunction in MPS I is unclear. It has been reported that neurons and glial cells accumulate GAG [1] and gangliosides [2]. Activation of glial cells [3] and alterations in oxidative status [4] in the cortex and cerebellum has been reported. It has been previously shown that MPS I mice had difficulty to habituate in the repeated open field test [5] and impaired long-term memory for aversive training [6]. Interestingly, although one study [7] reported abnormal performance of MPS I mice in Morris water maze tests, another study found inconclusive results [8]. In more recent studies, MPS I mice showed impaired learning behaviors in water T maze test [9] and spatial memory skills in the Barnes maze test [10]. These findings describe abnormal cognitive and neuropathology in MPS I, but the mechanisms are likely to be complex and remain to be elucidated.
Development of proteomics technology includes two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which has provided a powerful tool to study the complicated biological processes in cells and rapidly profiling the global protein expression alterations. 2D-PAGE is powerful in identifying proteins and protein isoforms that may be neglected by other methods such as in-solution digestion and nanoLC-MS/MS or 1D-PAGE and nanoLC-MS/MS. As a mass spectrometric screening method, 2D-PAGE offers many advantages: high throughput, broad dynamic range, good reproducibility and adequate sensitivity. Although combing 2D-PAGE with mass spectrometry is a common proteomic approach for high throughput screening of putative biomarkers in several disorders [11], [12], it has not been used in lysosomal diseases.
In this study, a comparative analysis of the proteome of MPS I and wildtype mouse brains using two dimensional gel electrophoresis (2D-GE) and nanoLC-ESI-MS/MS were performed. We identified 50 proteins that were differentially expressed in brains of MPS I versus wildtype mice. Bioinformatics analyses using Database for Annotation, Visualization, and Integrated Discovery (DAVID) [13], Protein ANalysis THrough Evolutionary Relationships (PANTHER) [14] and Search Tool for the Retrieval of Interacting genes (STRING) [15] databases allowed for functional classification of the detected proteins and highlighted MPS I-relevant biological pathways. This approach of screening identified potential biomarkers of MPS I that may reveal specific protein signatures indicating MPS I risk. These might also prove useful to assess prognosis, and provide outcome measures for assessing response to therapies.
Section snippets
Animals and sample collection
MPS I knockout mice (idua-/-), a kind gift from Dr. Elizabeth Neufeld, UCLA, has been generated by insertion of neomycin resistance gene into exon 6 of the 14-exon IDUA gene on the C57BL/6 background. MPS I mice (idua-/-) and wildtype were genotyped by PCR. All mouse care and handling procedures were in compliance with the rules of the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota. The whole mouse brains were collected from one MPS I and one wildtype mouse
2D-PAGE of brain samples from MPS I and wildtype mice
Brain samples from MPS I and wildtype mice (5-month old) were collected for identifying putative differentially expressed proteins using 2D-PAGE combined with nanoLC-LC-MS/MS. In all, 2 coomassie and 4 silver-stained (Fig. S1) gels of 2D-PAGE of the brain samples (250 μg protein in each gel) were run for reasons of reproducibility. A total of 2055 spots were compared (Fig. 1) and only those spots with a fold change ≥ 3.5 and a p value < 0.05 between MPS I and wildtype mice were picked for in-gel
Functional classification of dysregulated proteins
In this study, 50 proteins were revealed as differentially expressed between MPS I and wildtype mouse brains. These proteins function in diverse biological processes, and some functional groups are formed through cluster and pathway analysis. We discuss below the different pathways and their interdependency, as well as some interesting proteins.
Acknowledgements
The authors thank Kendrick Labs (Madison, WI, http://www.kendricklabs.com/) for 2D-PAGE and quantitative analyses. All authors declare that they have no conflicts of interest related to this work. This work is supported by NIH grant P01HD032652. Dr. Li Ou is a fellow of the Lysosomal Disease Network (U54NS065768). The Lysosomal Disease Network is a part of the Rare Diseases Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), and NxCATS. This
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