Chemyx: Analyzing Phosphorylation-State Changes in Neurodegenerative Disease with Mass Spectrometry

Subtle changes require a sensitive analysis method.

By | November 13, 2017

Disorder in the Brain: Proteins Gone Bad

Protein misfolding and aggregation is a hallmark of incurable neurodegenerative diseases like Alzheimer’s (AD), Parkinson’s (PD), and some dementia types. Immense efforts are underway to clarify the underlying mechanisms responsible, of which the consequences phosphorylation contributes remain incompletely understood. For example, the causative versus the consequential role of site-specific phosphorylation of aggregate-forming alpha-synuclein (aSyn) in PD continues to be contested.1,2 Moreover, hyperphosphorylation of the tau protein in AD and some types of dementia is known to drive its misfolding and aggregation, but the pathophysiological ramifications also continue to be challenged.2,3 

Therefore, in addition to physiologically-relevant in vivo models, accurate technical approaches are vital for the proper study of protein behavior, since this research primarily drives the development of novel biomarkers and therapeutics. To that end, mass spectrometry (MS) is utilized to precisely determine both the extent and location of phosphorylation present on protein(s)-of-interest, which together inspires their biological functions. 

Breaking Down Complex Neurodegenerative Disease with Mass Spectrometry

Researchers have two source options for MS analysis of in vivo samples: the cerebrospinal fluid (CSF) or post-mortem diseased brain tissue. The easily accessible and pathologically protein-rich CSF is especially useful in biomarker discovery, where its metabolic composition reflects that of the brain and can be compared with normal proteomic profiles.4

Already, MS-based approaches have uncovered phosphorylation sites on key disease-driving proteins and the acting kinases for potential therapeutic intervention.5-7 What’s more, upgrading with a phosphopeptide-enrichment step before MS analysis using metal oxide species (e.g., Fe(III)-IMAC, TiO2, ZrO2) makes phosphoproteomic analysis of pre-clinical samples or comprehensive proteomic screening more straightforward.4 The sample preparation and delivery process is streamlined via precise syringe pump-mediated infusion directly to the spectrometer. This automatic delivery of samples ensures accurate and reproducible sample analysis. 

Back Up: Why is Phosphorylation Important?

Protein phosphorylation is a form of post-translational modification that is instrumental to the basal regulation and maintenance of cellular protein function and turnover. At the flick of a switch, a protein’s catalytic activity is altered due to folding and conformational changes triggered by phosphorylation. The added phosphate group may also serve as a prerequisite signal for secondary modifications like ubiquitination, and thus degradation or transport to other organelles. Importantly, protein-protein interactions are also susceptible to manipulation by phosphorylation. These functional and positional fates of proteins are dictated by phosphorylation occurring within either the allosteric (regulatory) or orthosteric (active) site, where single or multiple residues can be targeted.8,9

Phosphorylation affects an array of proteins, either directly, by influencing protein folding patterns, or indirectly, via cross-talk with ubiquitination, impacting protein degradation. It is no surprise, then, that when the process goes awry, the biological implications are wide-ranging. Phosphorylation-dependent ubiquitination is a common prerequisite for many substrates of E3 ligases, which confer specificity to ubiquitin-proteasome system. Meaning, protein degradation can be both encouraged (maintenance) and inhibited (aggregation) via phosphorylation activity. Furthermore, hyperphosphorylation can prompt the misfolding and aggregation of proteins implicated in various diseases. These phenomena are central to the pathogenesis of the devastating diseases affecting the nervous system.

Ready When You Are: Direct Sample Infusion  

The use of MS in neurodegenerative proteomic studies is wide-spread but its capacity to accurately process and analyze samples hinges on the proper tuning and calibration of the spectrometer, followed by reliable direct infusion of samples for analysis. Direct syringe-associated infusion aids are a quick one-step solution to both automated calibration and reproducible data generated by MS-based methodologies.

References

1. D.F. Alonso, et al., “Excessive urokinase-type plasminogen activator activity in the euglobulin fraction of patients with Alzheimer-type dementia,” J Neurol Sci 139:83-88, 1996.

2. S. Tenreiro, et al., “Protein phosphorylation in neurodegeneration: friend or foe?” Front Mol Neurosci 7:1-30, 2014.

3. I. Grundke-Iqbal, et al., “Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology,” Proc Natl Acad Sci 83:4913-4917, 1986.

4. X. Wei and L. Li, “Mass spectrometry-based proteomics and peptidomics for biomarker discovery in neurodegenerative diseases,” Int J Clin Exp Pathol 2:132-148, 2009.

5. P. Davidsson and M. Sjogren, “The use of proteomics in biomarker discovery in neurodegenerative diseases, Dis Markers 21:81-92, 2005.

6. D.P. Hanger, et al., “New phosphorylation sites identified in hyperphosphorylated tau (paired helical filament-tau) from Alzheimer's disease brain using nanoelectrospray mass spectrometry,” J Neurochem 71:2465-2476, 1998.

7. P. Derkinderen, et al., “Tyrosine 394 is phosphorylated in Alzheimer's paired helical filament Tau and in fetal Tau with c-Abl as the candidate tyrosine kinase,” J Neurosci 25:6584-6593, 2005.

8. C. Salazar and T. Hofer, Multisite protein phosphorylation–from molecular mechanisms to kinetic models. FEBS J 276:3177-3198, 2009.

9. R. Nussinov, et al., “Allosteric posttranslational modification codes,” Trends Biochem Sci 37:447-455, 2012.

 

Meet the Sponsor: 

This article is brought to you by Chemyx, Inc. Syringe Pumps by Chemyx are used in top-level biomedical, pharmaceutical, chemical, and petrochemical research, offering highly precise, consistent, and reproducible fluidic delivery. Chemyx pump devices orchestrate the performance of different technologies that make modern research into novel materials, drugs, and energy resources possible. www.chemyx.com

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