Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product
Abstract
:1. Introduction
2. Results
2.1. Identification of AGEs Generated on the Model Protein MB
2.2. Absorption and Fluorescence Properties of MB-mel Products
2.3. Fourier-Transform Infrared Spectroscopy (FTIR) of the Glycated Myoglobin
2.4. Analysis of the Site-Specific Modifications Present on MB Glycated with Mel
2.5. MAGE Adducts Consisting of an Intact Melibiose
3. Discussion
4. Materials and Methods
4.1. Synthesis of Protein AGEs
4.2. SDS-PAGE and Western Blotting
4.3. Absorption Spectra
4.4. Fluorescence Spectra
4.5. Fourier-Transform Infrared Spectroscopy (FTIR)
4.6. Mass Spectrometry Analysis
4.7. Proteomic Data Analysis
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Type of Modification | Modified Residue | Detected m/z | ∆m [Da] | Score | Peptide Sequence |
---|---|---|---|---|---|
CML | K78 | 412.225 | +58.005 | 6.89 × 1014 | _K(CML)KGHHEAELK_ |
CML | K87 | 412.224 | +58.005 | 3.76 × 1014 | _KKGHHEAELK(CML)_ |
CML | K56 | 470.553 | +58.005 | 5.69 × 1014 | _TEAEMK(CML)ASEDLK_ |
CEL | K145 | 716.894 | +72.021 | 6.9 × 1014 | _ALELFRNDIAAK(CEL)_ |
CML | K63 | 522.318 | +58.005 | 1.97 × 1014 | _K(CML)HGTVVLTALGGILK_ |
Hex, CML 2× CEL | H64, K63, K62, K77 | 1257.686 | +364.100 | 5.35 × 1014 | _ASEDLK(CEL)K(CML)H(Hex)GTVVLTALGGILK(CEL)_ |
MGO | K56 | 469.222 | +54.010 | 4.69 × 1014 | _TEAEMK(MGO)ASEDLK_ |
Type of Modification | Modified Residue | Detected m/z | ∆m [Da] | Score | Peptide Sequence |
---|---|---|---|---|---|
2× Hex | K63, K77 | 611.018 | +324.106 | 7.45 × 1014 | _K(Hex)HGTVVLTALGGILK(Hex)_ |
3× MOLD | K63, K77, K78 | 594.690 | +147.024 | 3.97 × 1014 | _K(MOLD)HGTVVLTALGGILK(MOLD)K(MOLD)_ |
CML | K87 | 412.224 | +58.005 | 1.75 × 1014 | _KKGHHEAELK(CML)_ |
CML | K96 | 425.898 | +58.005 | 3.81 × 1014 | _PLAQSHATK(CML)HK_ |
CML | K56 | 470.553 | +58.005 | 8.26 × 1014 | _TEAEMK(CML)ASEDLK_ |
CML | K77 | 782.974 | +58.005 | 9.56 × 1014 | _HGTVVLTALGGILK(CML)K_ |
CML | K63 | 522.319 | +58.005 | 7.59 × 1014 | _K(CML)HGTVVLTALGGILK_ |
GAP | K78 | 539.325 | +109.029 | 7.48 × 1014 | _HGTVVLTALGGILKK(GAP)_ |
GOLD, DOGDIC | K78, K79 | 668.834 | +160.016 | 5.56 × 1014 | _K(GOLD)K(DOGDIC)GHHEAELK_ |
GOLD, DOGDIC | K87, K78 | 668.834 | +160.016 | 4.61 × 1014 | _K(DOGDIC)KGHHEAELK(GOLD)_ |
Hex | K62 | 476.747 | +162.053 | 5.48 × 1014 | _ASEDLK(Hex)K_ |
Hex | H93 | 611.018 | +162.053 | 4.7 × 1014 | _PLAQSH(Hex)ATKHKIPIK_ |
Hex | K98 | 611.017 | +162.053 | 4.31 × 1014 | _PLAQSHATKHK(Hex)IPIK_ |
Hex | K96 | 611.018 | +162.053 | 2.39 × 1014 | _PLAQSHATK(Hex)HKIPIK_ |
mel | R139 | 659.345 | +324.106 | 4.39 × 1014 | _ALELFR(mel)NDIAAKYK_ |
mel | K62 | 557.771 | +324.106 | 3.57 × 1013 | _ASEDLK(mel)K_ |
mel | K63 | 611.018 | +324.106 | 4.88 × 1014 | _K(mel)HGTVVLTALGGILK_ |
mel | H64 | 611.018 | +324.106 | 6.83 × 1014 | _H(mel)GTVVLTALGGILKK_ |
mel | K77 | 611.018 | +324.106 | 4.32 × 1014 | _KHGTVVLTALGGILK(mel)_ |
mel | K77 | 611.019 | +324.106 | 7.53 × 1014 | _HGTVVLTALGGILK(mel)K_ |
mel, 2× CML | H97, K96, K98 | 553.268 | +440.116 | 7.05 × 1014 | _PLAQSHATK(CML)H(mel)K(CML)_ |
MG-H1 | R139 | 569.313 | +54.010 | 4.32 × 1014 | _ALELFR(MG-H1)NDIAAKYK_ |
MG-H1 | R139 | 472.261 | +54.010 | 8.01 × 1014 | _ALELFR(MG-H1)NDIAAK_ |
Pyrraline | K96 | 593.008 | +108.021 | 1.18 × 1014 | _PLAQSHATK(Pyrraline)HKIPIK_ |
4× GA | H97, K98, H113, H116 | 1003.870 | +408.128 | 2.72 × 1014 | _H(GA)K(GA)IPIKYLEFISDAIIH(GA)VLH(GA)SK_ |
GLX | K96 | 496.762 | +39.995 | 1.18 × 1014 | _PLAQSHATK(GLX)_ |
GLX | H119 | 771.837 | +39.995 | 3.32 × 1014 | _H(GLX)PGDFGADAQGAMTK_ |
2× GLX | H93, K96 | 516.761 | +79.99 | 3.93 × 1014 | _PLAQSH(GLX)ATK(GLX)_ |
MGO | K56 | 469.224 | +54.010 | 4.34 × 1014 | _TEAEMK(MGO)ASEDLK_ |
MGO | K147 | 569.313 | +54.010 | 3.15 × 1014 | _ALELFRNDIAAKYK(MGO)_ |
2× MGO | K96, K102 | 593.008 | +108.020 | 1.18 × 1014 | _PLAQSHATK(MGO)HKIPIK(MGO)_ |
5× MGO | H93, K96, H97, K98, K102 | 647.021 | +270.050 | 1.15 × 1014 | _PLAQSH(MGO)ATK(MGO)H(MGO)K(MGO)IPIK(MGO)_ |
3× MGO | H93, K98, K102 | 611.010 | +162.030 | 3.62 × 1014 | _PLAQSH(MGO)ATKHK(MGO)IPIK(MGO)_ |
MGO | K145 | 472.261 | +54.010 | 5.68 × 1014 | _ALELFRNDIAAK(MGO)_ |
Type of Modification | Modified Residue | Detected m/z | ∆m [Da] | Score | Peptide Sequence |
---|---|---|---|---|---|
2× Hex | K56, K62 | 559.253 | +324.106 | 8.10428 × 1014 | _TEAEMK(Hex)ASEDLK(Hex)_ |
2× Hex, 2× CML | H64, K77, K78, K79 | 692.390 | +324.106 | 7.97853 × 1014 | _H(Hex)GTVVLTALGGILK(Hex)K(CML)K(CML)_ |
3× mel, CML, CEL | H81, H82, K87, K78, K79 | 760.337 | +1102.344 | 6.10181 × 1014 | _K(CML)K(CEL)GH(mel)H(mel)EAELK(mel)_ |
3× MOLD | K62, K63, K77 | 766.430 | +147.024 | 7.30365 × 1014 | _ASEDLK(MOLD)K(MOLD)HGTVVLTALGGILK(MOLD)_ |
CEL | K145 | 478.264 | +72.021 | 8.29719 × 1014 | _ALELFRNDIAAK(CEL)_ |
CEL | K77 | 725.934 | +72.021 | 3.53221 × 1014 | _HGTVVLTALGGILK(CEL)_ |
CML | K77 | 782.973 | +58.005 | 3.3215 × 1014 | _HGTVVLTALGGILK(CML)K_ |
CML | K145 | 570.644 | +58.005 | 6.11103 × 1014 | _ALELFRNDIAAK(CML)YK_ |
GOLD | K133 | 769.332 | +34.992 | 4.66304 × 1014 | _HPGDFGADAQGAMTK(GOLD)_ |
GOLD, DOGDIC | K87, K78 | 668.834 | +160.016 | 8.9105 × 1014 | _K(DOGDIC)KGHHEAELK(GOLD)_ |
Hex | K62 | 476.746 | +162.053 | 1.08165 × 1014 | _ASEDLK(Hex)K_ |
mel, 2× CML | H64, K63, K77 | 692.390 | +440.116 | 6.8908 × 1014 | _K(CML)H(mel)GTVVLTALGGILK(CML)K_ |
mel, CEL | K56, K62 | 583.261 | +396.127 | 5.48394 × 1014 | _TEAEMK(mel)ASEDLK(CEL)_ |
MOLD | K47 | 556.313 | +49.008 | 4.13698 × 1014 | _FDKFK(MOLD)HLK_ |
Pentosidine | K62 | 737.084 | +58.992 | 5.06918 × 1014 | _ASEDLK(Pentosidine)KHGTVVLTALGGILK_ |
2× GA | K62, K63 | 332.171 | +204.064 | 2.58266 × 1014 | _ASEDLK(GA)K(GA)_ |
4× GA | K79, H81, H82, K87 | 486.233 | +408.128 | 9.06642 × 1014 | _K(GA)GH(GA)H(GA)EAELK(GA)_ |
GLX | H64 | 709.921 | +39.995 | 3.29039 × 1014 | _H(GLX)GTVVLTALGGILK_ |
GLX | K77 | 473.617 | +39.995 | 8.98953 × 1014 | _HGTVVLTALGGILK(GLX)_ |
GLX | H113 | 642.341 | +39.995 | 8.29994 × 1014 | _YLEFISDAIIH(GLX)VLHSK_ |
MGO | K56 | 469.223 | +54.010 | 3.19781 × 1014 | _TEAEMK(MGO)ASEDLK_ |
Glycation Type | Residue (Amount within MB) | Residues with Intact Mel | % of Residue Type | % of Nucleophilic Residues (32 aa) | % of Total Residues (153 aa) |
---|---|---|---|---|---|
MWG | K (19) | 4 | 21.1 | 21.9 | 4.6 |
H (11) | 2 | 18.2 | |||
R (2) | 1 | 50.0 | |||
ACG | K (19) | 2 | 10.5 | 15.6 | 3.3 |
H (11) | 3 | 27.3 | |||
R (2) | 0 | 0.0 |
AGE Modification Type | Monoisotopic Mass [Da] [Da] [Da] |
---|---|
CML | 58.005 |
CEL | 72.021 |
Hex/FL | 162.053 |
Pyrraline | 108.021 |
Pentosidine | 58.992 |
GAP | 109.029 |
MG-H1 (MGO on R) | 54.010 |
GOLD | 34.992 |
MOLD | 49.008 |
DOGDIC | 125.024 |
mel | 324.106 |
GA | 102.032 |
GLX | 39.995 |
MGO (on K, H) | 54.010 |
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Gostomska-Pampuch, K.; Wiśniewski, J.R.; Sowiński, K.; Gruszecki, W.I.; Gamian, A.; Staniszewska, M. Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product. Int. J. Mol. Sci. 2022, 23, 13036. https://doi.org/10.3390/ijms232113036
Gostomska-Pampuch K, Wiśniewski JR, Sowiński K, Gruszecki WI, Gamian A, Staniszewska M. Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product. International Journal of Molecular Sciences. 2022; 23(21):13036. https://doi.org/10.3390/ijms232113036
Chicago/Turabian StyleGostomska-Pampuch, Kinga, Jacek R. Wiśniewski, Karol Sowiński, Wieslaw I. Gruszecki, Andrzej Gamian, and Magdalena Staniszewska. 2022. "Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product" International Journal of Molecular Sciences 23, no. 21: 13036. https://doi.org/10.3390/ijms232113036