MSACL 2017 US Abstract

Elucidation of Novel Proteoforms of Superoxide Dismutase (SOD1) in Sporadic ALS Patients

Philip Loziuk (Presenter)
NC State University

Bio: My background spans across many fields of untargeted and targeted quantitative mass spectrometry including: proteomics, post-translational modifications, metabolomics, glycomics, lipidomics, and TransOMICs which seeks to combine these different levels of information to provide a systems perspective of biology. I have studied a great deal of biological problems and systems including: lignin flux in trees, lipid biosynthetic pathways in neurodevelopment and more recently, in human clinical specimens for in-depth proteomic analysis of SOD1 in the context of ALS.

Authorship: Philip L. Loziuk, Lauren Nufer, Josh Pierce, Yasamin Moazami, Denis Fourches and David C. Muddiman,
North Carolina State University

Short Abstract

Mass spectrometry offers the most robust and specific platform to discover and characterize biomolecules. In the field of proteomics, there are three main approaches: top-down, middle-down, and bottom-up. This presentation will detail the characterization of SOD1 derived from patients diagnosed with sporadic ALS. The level of molecular detail (100% sequence coverage) using bottom-up proteomics allowed us to discover novel attributes of SOD1 including the incorporation of a nonprotein amino acid, novel nonsynonymous SNPs as well as low abundant PTM’s (<1% occupancy) pertaining to oxidative stress. New materials for the characterization and quantification of novel SOD1 proteoforms will also be presented. Finally, computational modeling of these proteoforms suggests that they have a significant impact on the structure of SOD1 and the progression of motor neuron diseases.

Long Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease which current impacts more than 12,000 people in the U.S. It is characterized by gradual motor neuron degeneration and motor neuron cell death, resulting in loss of voluntary muscle control. Currently, treatment is limited and diagnosis is performed by exclusion of other diseases, with no biochemical test available. Only about 5-10% of cases are familial or inherited with the remaining 90-95% being sporadic cases, occurring at random with no clearly associated risk factors. About 20% of familial cases result from mutations in the gene that encodes the enzyme copper-zinc superoxide dismutase 1 (SOD1). It has been demonstrated that SNPs and modifications of SOD1 including glutathionylation can cause dissociation of the native dimer, formation of trimers which are toxic to motor neurons [1-3]. Much work has also suggested that oxidative stress is highly associated with ALS, in particular lipid peroxidation and carbonylation [4-6]. There is also data that suggests exposure the toxic non-protein amino acid β-N-methylamino-L-alanine (BMAA) synthesized in cyanobacteria can result in motor neuron degeneration and that BMAA can be misincorporated into proteins during translation in vitro [7-11]. While much is known with respect to SOD1 modifications and SNPs, to date, an in-depth proteomic profile of SOD1 in humans diagnosed with sporadic ALS has remained largely unknown.

Purified SOD1 from human erythrocytes of 10 control and 7 patients diagnosed with ALS was obtained for UNC chapel hill school of medicine. Both intact and bottom-up approaches were employed. Samples were prepared by FASP [12]. Data was acquired by reversed phase nanoLC coupled to a QExactive High Field Mass Spectrometer utilizing data dependent acquisition. Intact data was deconvoluted using the Xtract algorithm. Bottom-up data was searched in proteome discoverer using closed (5ppm) searches for specific modifications and SNPs. Open searches were performed (500Da) to compile a list of potential unknown modifications present. Label-free quantitative data (NSpC and peak area) were inputted into a SMO machine learning algorithm to differentiate control and ALS patients. A SIL phosphoserine containing peptide unique to SOD1 was chemically converted (BMAA) using a Michael addition of methylamine, a scheme previously used for phosphopeptide labeling [13]. This peptide use utilized for PC-IDMS to identify incorporation of the toxic non-protein amino acid BMAA. Additionally, a novel SIL BMAA reagent synthesis provided a new useful tool for studying free BMAA and that from hydrolysates of biological samples. Computational microsecond timescale modeling of WT and modified SOD1 monomer and dimer were performed using PyMOL.

The most abundant proteoforms of SOD1 could be identified by Intact MS measurements including: acetylated, glutathionylated and phosphorylated. Phosphorylation occupancy was found to be differ significantly in ALS patients. In comparison, bottom-up data reduced the proteoform complexity and demonstrated the ability achieve greater depth, particularly with low abundant modifications. These data revealed 30 highly confident sites modified and 10 distinct modifications in SOD1, many related to ROS which haven’t been observed previously as well as 1 new SNP which has never been reported. Through label-free quantification using peak area and spectral counting, many of these modifications were found to differ between control and ALS groups. Further analysis utilizing machine learning revealed that combined opened and closed database searches provide >200 attributes in these data that sufficiently describe these groups, resulting in 100% correct classification when using half of the data as a training set. These methods will lead to better method of diagnosis and reveal potential avenues of intervention for therapeutics. PC-IDMS experiments revealed important findings regarding correctly identifying BMAA incorporation and important steps needed to avoid isobaric interferences and false positives. Computational modeling of 3 select modifications revealed significant disruption to the stability of the SOD1 dimer. Finally, we demonstrate a synthesis scheme for a new 13C315N2 SIL BMAA reagent which will provide a new analytical tool for identification and quantification BMAA.


References & Acknowledgements:

[1] McAlary L, Yerbury JJ, Aquilina JA, Glutathionylation potentiates benign superoxide dismutase 1 variants to the toxic forms associated with amyotrophic lateral sclerosis Sci Rep (2013) 3275.

[2] Shaw BF, Lelie HL, Durazo A, Nersissian AM, Xu G, Chan PK, Gralla EB, Tiwari A, Hayward LJ, Borchelt DR et al, Detergent-insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1 J Biol Chem 13 (2008) 8340-8350.

[3] Proctor EA, Fee L, Tao YZ, Redler RL, Fay JM, Zhang YL, Lv ZJ, Mercer IP, Deshmukh M, Lyubchenko YL et al, Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis P Natl Acad Sci USA 3 (2016) 614-619.

[4] Dasgupta A, Zheng JZ, Bizzozero OA, Protein carbonylation and aggregation precede neuronal apoptosis induced by partial glutathione depletion Asn Neuro 3 (2012).

[5] Sultana R, Perluigi M, Butterfield DA, Lipid peroxidation triggers neurodegeneration: A redox proteomics view into the Alzheimer disease brain Free Radical Bio Med (2013) 157-169.

[6] Curtis JM, Hahn WS, Long EK, Burrill JS, Arriaga EA, Bernlohr DA, Protein carbonylation and metabolic control systems Trends Endocrin Met 8 (2012) 399-406.

[7] Dunlop RA, Cox PA, Banack SA, Rodgers KJ, The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of L-Serine Causing Protein Misfolding and Aggregation Plos One 9 (2013).

[8] Cox PA, Davis DA, Mash DC, Metcalf JS, Banack SA, Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain P Roy Soc B-Biol Sci 1823 (2016).

[9] Torbick N, Hession S, Stommel E, Caller T, Mapping amyotrophic lateral sclerosis lake risk factors across northern New England Int J Health Geogr (2014).

[10] Stommel EW, Field NC, Caller TA, Aerosolization of cyanobacteria as a risk factor for amyotrophic lateral sclerosis Med Hypotheses 2 (2013) 142-145.

[11] Glover WB, Mash DC, Murch SJ, The natural non-protein amino acid N-beta-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis Amino Acids 11 (2014) 2553-2559.

[12] Loziuk PL, Wang J, Li QZ, Sederoff RR, Chiang VL, Muddiman DC, Understanding the Role of Proteolytic Digestion on Discovery and Targeted Proteomic Measurements Using Liquid Chromatography Tandem Mass Spectrometry and Design of Experiments J Proteome Res 12 (2013) 5820-5829.

[13] Adamczyk M, Gebler JC, Wu J, Identification of phosphopeptides by chemical modification with an isotopic tag and ion trap mass spectrometry Rapid Commun Mass Sp 10 (2002) 999-1001.


Financial Disclosure

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GrantsyesNIH, USDA, DOE,
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Stockno
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IP Royalty: no

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