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Optimization of ESI-QIT-TOF Mass Spectrometer and Cryogenic Ion Spectroscopy of Tryptic Peptide from Regulatory Loop of Insulin Receptor

  • 전효남 (Joen Hyo Nam, 아주대학교 일반대학원)

초록/요약

Cryogenic gas-phase spectroscopy provides high-resolution structural information by removing solvent interference and reducing thermal congestion, making it a powerful tool for analyzing selected segments of biomolecules. In a previous study, the tryptic peptide containing the two activation-loop tyrosines of Janus kinase 3 (DYYVVR) was investigated. The ππ* bands of each of the two tyrosine residues of DYYVVR were observed to be cleanly separated at cryogenic temperature. Motivated by this result, we sought to determine whether tyrosine residues from other kinase activation loops would likewise remain spectroscopically distinguishable in the gas phase. To pursue this goal, we constructed and optimized a custom electrospray ionization–quadrupole ion trap–time-of-flight (ESI– QIT–TOF) mass spectrometer designed for action spectroscopy. Vacuum performance was enhanced by installing an additional turbomolecular pump, and upstream CID fragments were selectively suppressed using a low-amplitude “tickle RF” applied to an ion guide. A closed-cycle cryostat cooled the ion trap to ~27 K, providing the conditions required for cryogenic analysis. We studied the tryptic peptide containing the three activation-loop tyrosines of the insulin receptor (DIYETDYYR). Room-temperature UV photodissociation (UVPD) spectra were broad but revealed a reproducible suppression of intensity for the Y1146-only peptide (DIYETDFFR) near 35 000–35 300 cm⁻¹, indicating heterogeneous π-solvation even at ambient temperature. At cryogenic temperature, H₂-tagging UVPD resolved three distinct ππ* bands: Y1146 produced the lowest-energy transition, while Y1150 and Y1151 contributed higher-energy, partially overlapping features. This result extends the residue-level separation observed previously in DYYVVR from two tyrosines to three and demonstrates that ππ* bands can serve as robust spectroscopic markers for individual sites in kinase activation loops. As a secondary objective, complementary H₂-tagging IR photodissociation (IRPD) and single- water-cluster UVPD measurements were performed to explore why the three tyrosines separate spectroscopically. These experiments established a solvation hierarchy in the phenolic O–H region: Y1150 exhibits the strongest OH-solvation, Y1151 intermediate OH-solvation, and Y1146 the weakest OH-solvation but the strongest π-solvation. Electronic-structure modeling supported this interpretation by reproducing the relative IR shifts. Although the mechanistic picture remains qualitative, these observations provide a plausible framework in which π-solvation stabilizes the Y1146 ππ* state, whereas OH-solvation dominates at the C-terminal sites. Taken together, this work demonstrates that DIYETDYYR can be spectroscopically resolved in the gas phase, establishing a direct extension of earlier two-residue separation and laying the foundation for generalizing tyrosine-selective cryogenic spectroscopy to additional kinase systems. The combination of custom instrumentation, cryogenic tagging methods, and solvation-based interpretation offers a versatile strategy for residue-level structural analysis in biomolecular fragments. Keywords Cryogenic ion spectroscopy; UVPD; IRPD; H₂ tagging; H₂O clusters; ESI–QIT–TOF; kinase activation loop; insulin receptor; tyrosine; π-solvation; OH-solvation; gas-phase photodissociation.

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목차

Chapter 1. Introduction 1
1.1 Background and Motivation 1
1.2 Objectives of the Thesis 5
1.3 Structure of the Thesis 6
Chapter 2. Principles and Previous Studies Related to Peptide Photodissociation 7
2.1 Spectroscopic Approaches to Biomolecular Structure Using Mass Spectrometry 7
2.2 Electrospray Ionization, Ion Guides, and Ion Trapping 9
2.2.1 Electrospray Ionization 9
2.2.2 Ion Guides and Quadrupole Ion Trap 10
2.2.3 Cryogenic Cooling of Trapped Ions 13
2.3 Action Spectroscopy in Gas-phase Biomolecules 14
2.4 Previous Research: Kinases and Insulin Receptor Activation Loops 16
2.4.1 Phosphorylation in Janus kinase 3 Activation Loops 16
2.4.2 Spectroscopic Studies on Janus kinase 3-derived Peptides 16
2.4.3 Extension to Insulin Receptor Activation Loop 18
Chapter 3. Experimental Setup 20
3.1 Overview of the home-made ESI-QIT-TOF Mass Spectrometer 20
3.2 Vacuum System 21
3.2.1 Existing Vacuum Configuration and Operating Pressures 21
3.2.2 Addition of a Turbo Pump for Enhanced Vacuum Performance 22
3.3 Ion Guides and Ion Lenses 26
3.3.1 Configuration of the Ion Guides and Lenses 26
3.3.2 Application of Tickle RF for Simplified Mass Filtering in the Ion Guide 34
3.3.3 Alignment–Efficiency Correlation in Adjacent Hexapole Guides 38
3.4 Ion Trap and TOF Components 39
3.5 Laser Systems 41
3.6 Cryogenic Ion Spectroscopy Setup 43
3.6.1 Cryogenic Cooler: Configuration and Cooling Mechanism 43
3.6.2 Copper Box and Connection to the Trap 45
3.6.3 Installation and Optimization of the Cooling System 51
3.7 Optimization of Photodissociation Experiments 55
3.7.1 UVPD of DYYVVR and Its derivatives at Room Temperature 55
3.7.2 Problems in Reproducing UVPD after System Changes 57
3.7.3 Trials of Photodissociation at Room Temperature 58
Chapter 4. Photodissociation Experiments of Tryptic Peptide from Regulatory Loop of Insulin Receptor 61
4.1 Background and Experimental Design 61
4.2 Photodissociation Spectroscopy at Room Temperature 62
4.2.1 Instrumental Configuration 62
4.2.2 Results of Room-Temperature UVPD 64
4.3 Photodissociation Spectroscopy under Cryogenic Conditions 66
4.3.1 Instrumental Configuration 66
4.3.2 Results of Cryogenic UVPD 68
4.3.3 Results of H₂-tagging UVPD & IRPD 70
4.3.4 Results of H₂O-cluster UVPD 73
4.4 Discussion 74
4.4.1 Structural Interpretation of Tryptic Peptide from Regulatory Loop of Insulin Receptor 74
4.4.2 Computational Results on Tryptic Peptide from Regulatory Loop of Insulin Receptor Structures 76
Chapter 5. Conclusion and Outlook 81
5.1 Summary of Findings 81
5.2 Future Directions 83
References 84

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