Changes in Protein Abundance by Age and Gender
Study 3 will support the signalling pathway analysis from study 2, by using a proteomic analysis of leukocytes across age and gender matched groups, to investigate differences in HC and PD participant protein abundance and gene expression along a specific signalling pathway.Abnormal Protein Expression and Neurodegenerative Progression
A key manifestation of neurodegenerative progression in PD pathogenesis is the abnormal expression and aggregation of proteins through transcriptional and translational responses to biological perturbations, resulting in post-translational modifications (Wetie, Woods, & Darie, 2014), altered protein–protein interactions, protein degradation and functional changes to the proteome (Dixit, Mehta, & Singh, 2019; Li, Ganz, & Smit, 2019), in disease progression (Rubinsztein, 2006). Impaired protein expression has been consistently associated with cellular damage, mitochondrial bioenergetic dysfunction, metabolic impairment, increased oxidative stress, elevated proinflammatory responses and increased apoptosis linked to neurodegenerative progression in PD (Dixit et al., 2019; Jiang et al., 2019; Kitsou et al., 2008; Licker, Kovari, Hochstrasser, & Burkhard, 2009). Identification of specific protein changes along an impaired bioenergetic signalling pathway offers the possibility of developing targeted pharmacological and clinical solutions to slowing and treating PD.
Proposed Procedures
Identification of changes in protein expression and abundance will use liquid chromatography (LC) to fractionate blood samples into similar protein groups (Zhang et al., 2012) then mass spectrometry (MS) will fragment each of these long chain proteins into smaller peptides (Chin et al., 2008; Zhang et al., 2012). The molecular weight of the proteins present in the sample will then be calculated and compared against pre-established databases for identification. Microarray/RNA-Sequencing will be used to compare changes in specific leukocyte derived pathway mean differential gene expression levels between HC and PD participants (Ihnatova & Budinska, 2015). Microarray/RNA-Seq data will be analysed using an R statistic routine within Bioconductor v3.2 software.
Proteomic Analysis Workflow. The workflow diagram shows PD and HC blood samples are collected and initially lysed to release cellular content into the cytosol. The sample is then centrifuged with the resulting supernatant submitted to a liquid chromatograph where the proteins are ionised and fractionated into similar protein groups. These protein groups are then passed through a mass spectrometer which fragments the long chain proteins into smaller protein peptides to enable the calculation of the molecular weight of each peptide. Software then compares the calculated molecular weight to a predefined protein database to identify the proteins present in the sample. Adapted from Plasma Proteome Profiling to detect and avoid sample-related biases in biomarker studies, by Philipp E Geyer. et.al., 2019. EMBO Molecular Medicine, 11(11), e10427. doi:10.15252/emmm.201910427.
Proteomic Differential Pathway Analysis
Differential protein comparisons between PD and HC participants by molecular weight (Zhang et al., 2012), and along mitochondria signalling pathways identified as impaired should reveal alterations in PD protein expression and abundance indicating the potential location of the mechanisms driving mitochondrial and protein impairment in PD. Understanding the changes in gene expression, protein alteration and the mitochondria mechanisms that are impaired will offer significant opportunities to target new clinical and pharmacological solutions in treating PD.
Proteomic Differential Pathway Analysis. Figure simulates an anticipated result for a proteomic differential pathway analysis comparing gene expressions between Parkinson disease participants and healthy controls along a specified pathway. (A) Differentially expressed OXPHOS protein heat map, (B) Protein-protein interaction analysis of the significantly differentially expressed proteins, and (c) Network map of impaired mitochondrial proteins. Adapted from A dose-dependent perturbation in cardiac energy metabolism is linked to radiation-induced ischemic heart disease in Mayak nuclear workers, by Azimzadeh, O., et.al. (2017), Oncotarget, 8(6), 9067-9078.