Biological Mass Spectrometry: Radical-Induced Protein Oxidation
From Fundamental Studies, Instrumentation and Bioanalytical to Biomedical Applications
Radicals of biomolecules are believed to play important roles in a variety of biological processes, including aging, stroke, and Parkinson’s disease, but their biochemical mechanisms are understood quite poorly at the molecular level. Our comprehensive studies have revolutionized our fundamental understanding of several hallmarks of radical-induced peptide chemistry—including the elementary processes of hydrogen atom, electron, and proton transfers, isomerizations, and dissociations—to shed light on the mechanisms of biological processes under oxidative stress. Our cross-disciplinary group is actively engaged in the fundamental, instrumental, and applied aspects of biological MS in advancing our fundamental understanding of radical-induced protein oxidation ranging from peptide radical chemistry as well as the development of new multi-dimensional liquid chromatography (MDLC)-MS instrumentation, technologies, methodologies for biomedical analysis and the mapping of molecular targets of downstream pathological processes; it stems from the fundamental research as well as development of MDLC-MS methodologies and instrumentation for both qualitative and quantitative proteomics, glycomics, and metabolomics analyses of complex biological samples in order to unravel radical-induced biochemical mechanisms under oxidative stress of a large number neurodegenerative disorders.
Biophysicals and Fundamental
The study of gas-phase peptide radical chemistry to form a scientific basis underlying peptide sequencing—one of the most important emerging bioanalyticl techniques used in proteomics applications—and to model charge/radical transfer in biological systems. Combinations of MS experiments and computational models have been used to examine the formation, isomerization, and dissociation of cationic and anionic peptide radicals through single electron transfer of transition metal/auxiliary ligand/peptide complexes under low-energy collision-induced dissociation, including the first demonstrations of the gas phase syntheses of a wide variety (dicationic, anionic, and aliphatic cationic) of radical peptides as well as site-specific isomeric ions with well-defined initial radical sites and post-translational modifications to reveal the electronic properties and reactivities of reactants, intermediates, transition state structures, and key products from the isomerizations and fragmentations steps. To gain new physical insight into radical peptide chemistry, we have also introduced laser-induced dissociation, ion trapping, and recently ion–molecule reactions within a hybrid linear ion trap tandem mass spectrometer. Our recent interdisciplinary approach combines collision-, surface-, electron- and laser-induced dissociation experiments with density functional theory and highly accurate time- and energy-resolved Rice–Ramsperger–Kassel–Marcus modeling to reveal the controlling factors behind the intriguing energetic and kinetic competitions among the various formation, isomerization and dissociation mechanisms.
Bioanalyticals and Next Generation MDLC-MS Instrumentation:
Complementary to our fundamental research studies, we have developed efficient technologies that have been proven in applications (see below) related to system biology-wide studies, as well as more-targeted studies on posttranslational modifications, including phosphorylation, glycosylation, and nitration. We are actively engaged in the development of new instrumentation and techniques for the bioanalytical applications of liquid chromatography/mass spectrometry. In collaboration with MDS-SCIEX and IONICS (Canada), we modify existing mass spectrometric hardware to explore new avenues for neuroproteomic, and metabolomic analyses. Our goals are to develop sensitive, efficient, and novel mass spectrometric methodologies. Online coupling of most MDLC systems without concomitant deterioration generally remains a challenge during conventional low-pH reversed-phase (RP) separation in the second dimension; thus, commercially available online MDLC systems have mainly been limited to combinations of 2D SCX-RP systems. Our recent studies into fully automated online MDLC/MS platforms with different degrees of analytical functionality and modes of separation—including the first examples of pH-10 RP/pH-2 RP, hydrophilic interaction chromatography (HILIC)–RP, and porous graphitic carbon chromatography (PGC)-RP MDLC technologies to accelerate qualitative and quantitative proteomics analyses, as well as recent implementations of our MDLC technologies into commercial off-the-shelf instruments; these approaches will allow biomedical researchers to adopt the technologies in a simple manner for unattended, robust, and high-throughput analyses of samples on the sub-microgram scale.
Biomedical Applications and Drug Discovery:
In our laboratory we are using our state-of-the-art technologies to investigate the ensemble of proteins expressed, and also the differences among them, in several challenging neurodegenerative diseases (Parkinson, ischemia stroke, and aging) and in the pharmacological neuroprotective effects of the associated process; such studies have been an integral part of our interdisciplinary biomedical research. Our previous and ongoing investigations into the molecular discovery of biomolecule oxidation in radical-induced diseases—stemming from our fundamental studies to biophysical, and bioanalytical applications—have been amalgamated with particular emphasis on reactive nitrogen/oxygen species–mediated modification of proteins such as 3-nitrotyrosine protein modification: a permanent and hallmark biomarker underlying molecular mechanisms of radical-induced pathological processes. In collaborations with biologists and biomedical scientists, we have made advances, using cell and animal models, to isolate signaling pathways that are deregulated, potentially informing strategies and targets for therapeutics. The advent of multiple drug therapy in traditional Chinese medicine offers new hope in addressing these complex pathological aspects by combining drug molecules with different modes of action, acting on multiple malfunctioning targets and biological processes that cause the chronic and progressive neurodegeneration disorders. We used polypharmacology-based strategies to examine the combined neuroprotective effects of new multi-target chemical components, identified and extracted from promising candidate herb(s). Our findings suggest that pharmacological interaction of polyphenol compounds in combination exhibits its enhanced neuroprotective effects through combination of cellular mechanisms: antioxidant cytoprotection and anti-inflammation for treating diseases such as Parkinson’s disease.
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A B C D
A: Immunohistochemical analysis of endothelial cells;
B: A novel metabolic gene in sorghum and zebrafish embryos;
C: MRI image of DDD (Degenerative Disc Disease);
D: MRI image of Macaca
brain (Degenerative disorder)
1. Zhang Z, Li G, Szeto SSW, Lee SMY, Chu IK. Free Radic. Biol. Med.. 2015, in press.
2. Zhao Y, Szeto SSW, Kong PW, Law CH, Li G, Quan Q, Zhang Z, Chu IK. Anal. Chem. 2014, 86 (24), 12172-12179.
3. Quan Q, Song T, Hao Q, Siu CK, Chu IK. J. Am. Soc. Mass Spectrom. 2013, 24 (4), 554-562.
4. Kong PW, Hao Q, Lai CK, Siu CK, Chu IK, J. Am. Soc. Mass Spectrom. 2012, 23 (12), 2094-2101.
5. Song, T.; Hao, Q.; Law, C. H.; Siu, C. K.; Chu IK. J. Am. Soc. Mass Spectrom. 2012, 23 (2), 264-273.
6. Kong PW, Siu SO, Lee SMY, Lo C, Chu IK. J. Chromatogr. A. 2011, 1218 (23), 3681-3688.
7. Siu SO, Lam MPY, Lau E, Kong PW, Zhang J, Lee SMY, Chu IK. Proteomics. 2011, 11(11), 2308-2319.
8. Laskin J, Yang ZB, Song T, Lam CNW, Chu IK. J. Am, Chem. Soc. 2010, 132 (45), 16006-16016.
9. Laskin J, Yang Z, Chu IK. J. Am. Chem. Soc. 2008, 130, 3218-3230.
10. Chu IK, Zhao J, Xu M, Siu SO, Hopkinson AC, Siu KWM. J. Am. Chem. Soc. 2008, 130, 7862-7872.
11. Laskin J, Futrell JH, Chu IK. J. Am. Chem. Soc. 2007, 129, 9598-9599.
12. Laskin J, Yang ZB, Lam C, Chu IK. Anal. Chem. 2007, 79, 6607-6614.
Invited book chapter and review article:
1. Lam MPY, Law CH, Quan Q, Zhao Y, Chu IK, Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, 2014, vol.1156,
2. Chu IK, Laskin J, Formation of peptide radical ions through dissociative electron transfer in ternary metal-ligand-peptide complex. Eur. J. Mass Spectrom. 2011, 17 (6), 543-556.
Recent event: Mass Spectrometry and Proteomics Workshop 2014: The Arts and Science of Mass Spectrometry — From Fundamentals to Applications
To foster a better research atmosphere and graduate student training on MS fundamental and applied research in Asia, we are so excited to share the announcement and officially launch of the Trent MS Conference - Asian Chapter during the symposium. More details are coming soon.