N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. hTSH Preparations
4.2. In Vivo Bioassay
4.3. Immunoradiometric Assay (IRMA)
4.4. Pharmacokinetic Studies
4.5. Mass Spectrometry for Molecular Mass Determination
4.6. N-Glycosylation Profiling by MALDI-TOF-MS
4.6.1. Glycosidase Digestion and Permethylation of N-Glycans
4.6.2. MALDI-TOF Analysis of N-Glycans
4.6.3. Average N-Glycan Mass and Monosaccharide Molar Ratio Determination on the Basis of Glycoprofiling
4.7. Site-Occupancy and Mass of the Carbohydrate Moiety Determination
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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N-Glycan 1 | Underivatized Mass (-H2O) (Da) | Relative Intensity of Each N-Glycan per Each Preparation (%) | ||||
---|---|---|---|---|---|---|
P1 | P2 | R1 | R2 | R3 2 | ||
(1) 0 | 892.3 | 2.0 | ||||
(2) F1 | 1038.4 | 1.9 | ||||
(3) M1 | 1054.4 | 3.7 | ||||
(4) M2 | 1216.4 | 7.0 | ||||
(5) N1F1 | 1241.5 | 2.7 | ||||
(6) N1G1 | 1257.5 | 1.3 | ||||
(7) N2 | 1298.5 | 0.4 | 0.8 | |||
(8) N1Gn1(SO4) | 1378.4 | 2.0 | 5.1 | |||
(9) M3 | 1378.5 | 9.0 | ||||
(10) M1N1G1/M2N1 | 1419.5 | 0.9 | ||||
(11) N2F1 | 1444.5 | 3.2 | 0.3 | 0.7 | ||
(12) N2G1 | 1460.5 | 1.9 | 0.4 | 0.4 | 1.9 | 1.4 |
(13) N1Gn1F1(SO4) | 1524.5 | 0.8 | 1.5 | |||
(14) N1G1 Gn1(SO4) | 1540.5 | 2.2 | 5.2 | |||
(15) M4 | 1540.5 | 1.5 | ||||
(16) N1G1S1 | 1548.5 | 8.2 | 1.8 | |||
(17) N2Gn1(SO4) | 1581.5 | 2.1 | 6.7 | |||
(18) M2N1G1/N1M3 | 1581.6 | 0.8 | ||||
(19) N2G1F1 | 1606.6 | 2.4 | 1.0 | 1.9 | ||
(20) N2G2 | 1622.6 | 2.1 | 5.0 | 6.2 | 3.8 | |
(21) N3F1 | 1647.6 | 2.0 | 1.8 | |||
(22) M1N1Gn1F1(SO4) | 1686.6 | 0.7 | ||||
(23) N1G1S1F1 | 1694.6 | 4.1 | 1.1 | |||
(24) M2N1Gn1(SO4) | 1702.5 | 0.3 | 2.5 | |||
(25) M1N1G1S1 | 1710.6 | 1.2 | 1.5 | |||
(26) N2Gn1F1(SO4) | 1727.6 | 1.4 | ||||
(27) N2G1Gn1(SO4) | 1743.6 | 1.8 | 3.6 | |||
(28) N2G1S1 | 1751.6 | 1.3 | 0.5 | 3.1 | 12.3 | |
(29) N2G2F1 | 1768.6 | 0.6 | 0.6 | 1.6 | ||
(30) N2Gn2(SO4) | 1784.6 | 0.6 | ||||
(31) N2G1Gn1F1 | 1809.7 | 1.5 | 0.8 | |||
(32) N2Gn2(SO4)2 | 1864.6 | 3.4 | ||||
(33) N2G1Gn1F1(SO4) | 1889.6 | 4.6 | ||||
(34) N2G1S1F1 | 1897.7 | 2.7 | 1.0 | |||
(35) N2G2S1 | 1913.7 | 1.9 | 0.9 | 36.1 | 26.3 | 21.4 |
(36) N2Gn2F1(SO4) | 1930.7 | 0.3 | 1.6 | |||
(37) N2G1Gn1F2 | 1955.7 | 1.8 | 0.8 | |||
(38) N2G1Gn1S1 | 1970.7 | 1.6 | 0.7 | |||
(39) N3G3 | 1987.7 | 2.4 | ||||
(40) N2Gn2F1(SO4)2 | 2010.6 | 0.6 | 4.4 | |||
(41) N3G2Gn1 | 2028.7 | 1.3 | ||||
(42) N2G1Gn1S1(SO4) | 2034.7 | 14.0 | ||||
(43) N2G2S1F1 | 2059.7 | 1.3 | 3.4 | 4.1 | 11.0 | |
(44) N2G1Gn1S1F1 | 2100.7 | 5.5 | 4.2 | |||
(45) N3G2F2 | 2117.8 | 0.5 | 1.0 | |||
(46) N3G2Gn1F1 | 2174.8 | 1.4 | ||||
(47) N2G1Gn1S1F1(SO4) | 2180.7 | 3.5 | ||||
(48) N2G2S2 | 2204.8 | 10.0 | 14.1 | 31.9 | 31.2 | 0.6 |
(49) N2G1Gn1S2 | 2245.8 | 2.6 | 2.3 | |||
(50) N3G3S1 | 2278.8 | 2.7 | 3.7 | |||
(51) N2G2S2F1 | 2350.8 | 1.9 | 2.1 | 5.3 | 4.8 | 1.5 |
(52) N3G3S1F1 | 2424.9 | 0.5 | 1.1 | |||
(53) N4G4F1 | 2498.9 | 1.0 | ||||
(54) N3G2S2F1 | 2553.9 | 1.3 | 0.5 | |||
(55) N3G3S2 | 2569.9 | 6.9 | 7.7 | 1.8 | ||
(56) N4G3Gn1S1 | 2685.0 | 0.8 | ||||
(57) N3G3S2F1 | 2716.0 | 1.5 | 1.6 | |||
(58) N4G3Gn1S1F1 | 2831.0 | 1.0 | ||||
(59) N3G3S3 | 2861.0 | 0.6 | 3.5 | 4.9 | 5.0 | |
(60) N4G4S2 | 2935.0 | 2.6 | ||||
(61) N3G3S3F1 | 3007.1 | 1.4 | 0.9 | 0.7 | 0.9 | 9.5 |
(62) N4G4S3 | 3226.1 | 3.2 | ||||
(63) N4G4S4 | 3517.2 | 6.7 | ||||
(64) N4G4S4F1 | 3663.3 | 10.7 | ||||
Fucosylated glycans | 36.0 | 35.2 | 11.9 | 13.5 | 37.9 | |
Sialylated | 41.2 | 50.8 | 94.2 | 90.9 | 86.3 | |
Sulfated | 13.7 | 55.7 | - | - | - | |
Bi-antennary | 46.8 | 73.7 | 82.6 | 77.6 | 57.0 | |
Triantennary | 4.7 | 6.5 | 16.5 | 20.9 | 18.7 | |
Tetraantennary | - | 1.8 | - | - | 24.2 |
hTSH Preparation | Molecular Mass by MALDI-TOF-MS (Da) | Average Glycan Mass (Da) | Monosaccharide or Sulfate Moles/hTSH Mole | Carbohydrate Moiety (%) | Occupancy Occupied Sites | (%) | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Fuc | GalNAc | GlcNAc | Gal | Man | SA | Sulfate | |||||||
P-1 | 27,611 | 1670.5 | 0.71 | 0.45 | 6.51 | 1.39 | 7.57 | 0.96 | 0.23 | 11.88 1 | (11.2) 2 | 1.96/3 | 65.3 |
P-2 | 28,524 | 1931.4 | 0.77 | 1.84 | 8.59 | 1.98 | 6.84 | 1.41 | 1.54 | 14.70 1 | (14.3) 2 | 2.17/3 | 72.3 |
R-1 | 29,921 | 2128.2 | 0.30 | 0 | 11.04 | 5.63 | 8.08 | 3.76 | 0 | 18.68 1 | (18.0) 2 | 2.63/3 3 | 87.7 |
R-2 | 29,085 | 2137.6 | 0.30 | 0 | 9.43 | 4.72 | 6.85 | 3.13 | 0 | 16.35 1 | (16.5) 2 | 2.22/3 | 74.0 |
R-3 | 29,281 | 2416.2 | 0.74 | 0 | 9.93 | 4.92 | 6.67 | 3.25 | 0 | 16.91 1 | (16.5) 2 | 2.05/3 | 68.3 |
Preparation | T4 Level (µg/dL) |
---|---|
P-1 | 1.71 ± 0.13 |
P-2 | 2.37 ± 0.64 |
R-1 | 2.85 ± 0.34 |
R-2 | 3.05 ± 0.70 |
R-3 | 2.57 ± 0.64 |
Preparation | No. of Assays | t1/2 1 (min) | Signif. Level | AUC 2 (µg∙min/mL) | Signif. 3 Level |
---|---|---|---|---|---|
P-1 | 2 | 56.5 ± 15.7 | p < 0.05 | 11,463 ± 682.0 | p < 0.05 |
P-2 | 2 | 89.7 ± 0.42 | p < 0.05 | 14,270 ± 2147.1 | NS |
R-1 | 3 | 115.1 ± 3.12 | - | 16,155 ± 1398.1 | - |
R-2 | 3 | 105.5 ± 18.4 | NS | 15,555 ± 1197.8 | NS |
R-3 | 3 | 114.5 ± 26.9 | NS | 16,976 ± 1720.0 | NS |
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Ribela, M.T.C.P.; Damiani, R.; Silva, F.D.; Lima, E.R.; Oliveira, J.E.; Peroni, C.N.; Torjesen, P.A.; Soares, C.R.; Bartolini, P. N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins. Int. J. Mol. Sci. 2017, 18, 131. https://doi.org/10.3390/ijms18020131
Ribela MTCP, Damiani R, Silva FD, Lima ER, Oliveira JE, Peroni CN, Torjesen PA, Soares CR, Bartolini P. N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins. International Journal of Molecular Sciences. 2017; 18(2):131. https://doi.org/10.3390/ijms18020131
Chicago/Turabian StyleRibela, Maria Teresa C. P., Renata Damiani, Felipe D. Silva, Eliana R. Lima, João E. Oliveira, Cibele N. Peroni, Peter A. Torjesen, Carlos R. Soares, and Paolo Bartolini. 2017. "N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins" International Journal of Molecular Sciences 18, no. 2: 131. https://doi.org/10.3390/ijms18020131