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Development of a novel quadrupole ion trap time-of-flight mass spectrometer and cryogenic ion spectroscopy for identification of phosphotyrosine

  • 권장한 (아주대학교 에너지시스템학과 응용화학전공)

초록/요약

We constructed a novel QIT-TOF mass spectrometer. We obtained room temperature UV PD spectra at the home of singly protonated tyrosine and singly protonated hexapeptides, DYYVVR. DYYVVR is a tryptic peptide of Janus kinase 3 and has the two active tyrosine residues needed to regulate of the protein’s activity by phosphorylation. Although we obtained a well-resolved spectrum at room temperature for singly protonated tyrosine, two adjacent tyrosine absorption spectra in singly protonated DYYVVR were not distinguishable. Therefore, cryogenic ion spectroscopy (CIS) was used to elucidate the spectrum results of singly protonated DYYVVR at room temperature. Through comparison with point mutants, it was determined that the lower electronic band to the absorption of 981st tyrosine, while the higher band to 980th tyrosine. In the case of phosphorylation, the UV absorption of the phosphorylated chromophore shifts to higher energy above 36500 cm–1. In contrast, the unphosphorylated chromophore retains its absorption in the same region. It is proved that CIS can be used to identify the two adjacent tyrosine chromophores and to confirm the phosphorylation site of a kinase homology domain. In the case of room temperature, UVPD spectra are also used to differentiate the absorption of two adjacent tyrosine chromophores. Mass spectrometry and ultraviolet spectroscopy in 2-dimensional analysis can effectively demonstrate to identify the location of phosphorylation sites in a peptide.

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목차

PART Ⅰ DEVELOPMENT OF A NOVEL QUADRUPOLE ION TRAP TIME-OF-FLIGHT MASS SPECTROMETER 1
I. INTRODUCTION 2
1.1 Methods of structural determination 2
1.2 Laser spectroscopy in biomolecules 3
1.3 Gas phase spectroscopy in biomolecules 4
1.4 Cryogenic temperature in biomolecules 5
II. DESCRIPTION AND CHARACTERIZATION OF A NOVEL QUADRUPOLE ION TRAP TIME-OF-FLIGHT MASS SPECTROMETER 7
2.1 APPARATUS OVERVIEW 7
2.1.1 Electrospray ionization 7
2.1.2 Vacuum chamber 10
2.1.3 Hexapole ion guides 12
2.1.4 Quadrupole ion trap 14
2.2 OPTIMIZATION OF THE APPARATUS 22
2.2.1 Condition of voltage 22
2.2.2 Ion trapping and He injection time 24
2.3 LASER SETUP FOR ULTRAVIOLET PHOTO DISSOCIATION 26
2.3.1 Generation of UV laser beam 26
2.3.2 spectroscopic technique schemes 27
III. MASS SPECTRA OF TYRH+ AND DYYVVRH+ 30
3.1 MASS SPECTRA OF TYRH+ 30
3.2 COLLISION-INDUCED DISSOCIATION OF TYRH+ 32
3.3 MASS SPECTRUM OF DYYVVRH+ 33
IV. PHOTO DISSOCIATION OF TYRH+ AND DYYVVRH+ 34
4.1 UV Photo dissociation of TyrH+ 34
4.2 UV PHOTO DISSOCIATION OF DYYVVRH+ 36
4.3 UV PHOTO DISSOCIATION OF DYFVVRH+ 37
4.4 UV PHOTO DISSOCIATION OF DFYVVRH+ 38
V. CONCLUSION 40
PART Ⅱ CRYOGENIC ION SPECTROSCOPY OF A SINGLY PROTONATED PEPTIDE DYYVVR: LOCATING PHOSPHORYLATION SITES OF A KINASE DOMAIN 41
I. INTRODUCTION 42
1.1 POST TRANSLATIONAL MODIFICATIONS 42
1.1.1 Phosphorylation 43
1.1.2 Acetylation 43
1.1.3 Ubiquitylation 44
1.1.4 Methylation 45
1.1.5 Glycosylation 46
1.1.6 SUMOylation 47
1.1.7 Palmitoylation 47
1.2 JANUS KINASE FAMILY 49
1.3 TRYPTIC HEXAPEPTIDES: DYYVVR, DYFVVR, DFYVVR, AND PHOSPHORYLATED DYYVVR IN THE ACTIVATION LOOP OF JAK3 50
II. EXPERIMENTAL METHOD 53
2.1 APPARATUS PART 53
2.2 CALCULATION PART 55
III. RESULT AND DISCUSSION 56
3.1 UVPD SPECTROSCOPY OF SINGLY PROTONATED PEPTIDES 56
3.1.1 UVPD spectrum of singly protonated DYYVVR 56
3.1.2 UVPD spectrum of singly protonated DYFVVR 58
3.1.3 UVPD spectrum of singly protonated DFYVVR 59
3.1.4 UVPD spectrum of singly protonated DpYYVVR 60
3.1.5 UVPD spectrum of singly protonated DYpYVVR 61
3.1.6 UVPD spectrum of singly protonated DpYpYVVR 62
3.2 UVPD SPECTROSCOPY OF DOUBLY PROTONATED PEPTIDES 64
3.2.1 UVPD spectrum of doubly protonated DYYVVR 64
3.2.2 UVPD spectrum of doubly protonated DYFVVR 65
3.2.3 UVPD spectrum of doubly protonated DFYVVR 66
3.3 IR-UV DOUBLE RESONANCE SPECTROSCOPY OF SINGLY PROTONATED PEPTIDES 67
3.3.1 IR-UV dip spectra of singly protonated DYYVVR 67
3.3.2 IR-UV dip spectra of singly protonated DFYVVR 69
3.3.3 IR-UV dip spectra of singly protonated DpYYVVR 70
3.3.4 IR-UV dip spectra of singly protonated DYpYVVR 71
3.4 H2 TAGGING SPECTROSCOPY OF SINGLY PROTONATED PEPTIDES 73
3.4.1 H2 tagging spectrum of singly protonated DYYVVR 73
3.4.2 H2 tagging spectrum of singly protonated DYFVVR 75
3.4.3 H2 tagging spectrum of singly protonated DFYVVR 76
3.5 DFT CALCULATION 77
IV. CONCLUSION 81
REFERENCE 82

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