simple and quick ms analysis of chemically synthesized … · 2017. 6. 16. · simple and quick ms...

Simple and Quick MS Analysis of Chemically Synthesized Glycopeptides: Hydrogen/Deuterium Exchange (HDX)/MS for Verifying Racemization *Izumi Sakamoto (1), Kenji Hirose (2)

Introduction

(1) AcroScale Inc., Sendai, Japan; (2) Nihon Waters K.K., Osaka, Japan

The production of glycoproteins is facing a new era. Chemical syntheses of

biologically active glycoproteins such as erythropoietin (EPO) and interferon

beta (IFN-b) have recently been realized. Since such processes are often

accompanied by racemizations of amino acid residues and chromatographic

separation of the resulting diastereomeric peptides is practically difficult, a

simple and quick detection method is highly required. Here, we present the use

of hydrogen/deuterium exchange (HDX) / MS to reveal the partial racemization

of amino acid residues in chemical syntheses of glycopeptides.

Concept & Methods

Asn

The chemical synthesis of sialylglycoproteins is challenging because of the unexpected lability of the sialyl

linkage under acidic conditions, which is a common problem in organic synthesis

R = H R = Protective

Group

Interferon b-1a

• Glycoprotein, 166 amino acid residues

• Glycosylated at Asn80

Lability of the sialyl linkage

under acidic conditions

Using the protective group

Deprotection : Hydrolysis with sodium hydroxide Racemization of amino acid residues?

A glycopeptide was treated with sodium deuteroxide solution for deprotection. After treatment of the

reaction mixture with H2O, hydrogen/deuterium exchange (HDX) via enolization of the carbonyl Cα-H

was monitored by ESI-QTOF-MS.

Monitoring Q-TOF systems

NaOD

D2O

OR

Desirable Condition

Undesirable Condition

Results

Chemical Synthesis of Glycopeptides / Glycoproteins

0.1 mM

Sample

50 mM NaOD

D2O,

Temp., Time

Sample Interferon β fragment

Chromatogram (TIC):

Temp.: 0 ℃,

Reaction Time: 30 min, 60 min, 180 min

Chromatogram

After deprotection, the peak remained at the same retention time.

12/09/12 2238-24-Bn on ice 180min 10uL injection

Time6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30

%

3

6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30

%

1

6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30

%

1

6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30

%

2

IS_2238_24_Bn_ice_180min 1: TOF MS ES+ TIC

3.72e4

6.881544.69

6.07500.88


4.13e4

6.881544.64

6.12500.90


4.03e4

6.881544.32

6.08541.93

IS_2238_24_Bn_ice_start 1: TOF MS ES+ TIC

3.05e4

7.551604.35

7.291574.347.19

1574.00

6.901543.98

6.02524.91

Synapt12/09/12 2238-24-Bn on ice 180min 10uL injection

m/z1156 1157 1158 1159 1160 1161 1162 1163 1164 1165

%

0

100

m/z1156 1157 1158 1159 1160 1161 1162 1163 1164 1165

%

0

100

m/z1156 1157 1158 1159 1160 1161 1162 1163 1164 1165

%

0

100

IS_2238_24_Bn_ice_180min 796 (6.889) Sm (SG, 2x2.00); Cm (792:802) 1: TOF MS ES+ 544

A: 4627.00±0.001158.75

1158.49

1158.24

1157.99

1159.00

1159.25

1159.50

1159.75

1160.01

1160.251160.48 1160.77

1161.08 1161.60


A: 4627.00±0.001158.75

1158.49

1158.23

1157.99

1159.01

1159.26

1159.51

1159.74

1160.001160.25 1160.47


A: 4627.00±0.001158.49

1158.23

1157.99

1158.75

1159.00

1159.25

1159.50

1159.76

1159.99 1160.271161.16

Spectrum: Calcd.:[M+3H]3+1157.98

Start

30 min

60 min

180 min

Temp.: 0 ℃, 24 ℃, 50 ℃

Reaction Time: 60 min

12/09/12 2238-24-Bn on ice 60min 10uL injection

Time6.00 6.05 6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 6.55 6.60 6.65 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 7.15 7.20 7.25 7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.95 8.00 8.05 8.10 8.15 8.20 8.25 8.30

%

2

6.00 6.05 6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 6.55 6.60 6.65 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 7.15 7.20 7.25 7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.95 8.00 8.05 8.10 8.15 8.20 8.25 8.30

%

3

6.00 6.05 6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 6.55 6.60 6.65 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 7.15 7.20 7.25 7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.95 8.00 8.05 8.10 8.15 8.20 8.25 8.30

%

7

IS_2238_24_Bn_50_60min 1: TOF MS ES+ TIC

1.33e4

6.911548.38

6.701548.37

6.591548.35

6.361548.31

5.97500.90

7.89523.93

IS_2238_24_Bn_rt_60min 1: TOF MS ES+ TIC

2.90e4

6.891546.35

6.721546.65

6.09500.90


4.22e4

6.881544.64

Thz-IFRQDSSSTGWN(R1)ETIVENLL-OH,

R1 = protected oligosaccharide

Synapt12/09/12 2238-24-Bn on ice 60min 10uL injection

m/z1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169

%

0

100

m/z1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169

%

0

100

m/z1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169

%

0

100

IS_2238_24_Bn_50_60min 798 (6.906) Sm (SG, 2x2.00); Cm (792:803) 1: TOF MS ES+ 1651161.52

1161.28

1161.02

1160.78

1160.50

1160.29

1156.751156.54

1161.79

1162.01

1162.28

1162.54

1162.79

1163.02

1164.721163.49

1164.981165.591166.23

IS_2238_24_Bn_rt_60min 796 (6.889) Sm (SG, 2x2.00); Cm (792:804) 1: TOF MS ES+ 450

A: 4627.00±0.001159.75

1159.51

1159.26

1159.01

1158.76

1158.48

1160.01

1160.27

1160.53

1160.76

1161.03

1161.27

1162.45 1163.18


A: 4627.00±0.001158.73

1158.49

1158.23

1157.99

1159.00

1159.26

1159.51

1159.74

1160.00

1160.47

Chromatogram:

The peak remained at the same

retention time.

0 ℃

24 ℃

50 ℃

The reaction conditions for the removal of the benzyl groups were

selected according to this monitoring. (Temp.: 0 ℃, Time: ～ 60 min)

Glycopeptide : Evaluation of reaction conditions

1.50 mM NaOD,

D2O, Temp.,60 min

2. Lyophilization

(with H2O), Twice

Sample Interferon β fragment

Thz-IFRQDSSSTGWN(R1)ETIVENLL-OH,

R1 = protected oligosaccharide

Glycopeptide : Monitoring with deuterium label

Endo-M,

0.1 mM PB (pH 6.0),

35 ℃, 90 min

Temp.: 0 ℃

Synapt12/09/20 2238-40 on ice EndoM (+) 10uL injection

m/z1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330

%

0

100

IS_2238_40_ice_with_EndoM 870 (7.528) Sm (SG, 2x2.00); Cm (867:876) 1: TOF MS ES+ 2.38e31315.65

1315.14

1314.65

1314.15

1316.16

1316.67

1317.18

1317.69

1318.18

1318.69

Synapt12/09/20 2238-40 rt EndoM (+) 10uL injection

m/z1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330

%

0

100

IS_2238_40_rt_with_EndoM 872 (7.545) Sm (SG, 2x2.00); Cm (867:878) 1: TOF MS ES+ 1.25e31317.18

1316.67

1316.16

1315.67

1315.16

1317.67

1318.18

1318.69

1319.20

1319.69

1320.21

1320.70

Spectrum :

Calcd: [M+2H]2+ 1314.11

Found: [M+2H]2+ 1314.15

Spectrum :

Calcd: [M+2H]2+ 1314.11

Found: [M+2H]2+ 1315.16

Part of the peptide fragment labeled with deuterium

Glycoprotein

Sample Interferon β

sialyl(Bn)-IFN b-1a sialyl-IFN b-1a

ASS-006-029-6 24 degree 60min 1796-33 0.1%TFA BEH130 C18 #186003556 (0228341401)

m/z750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950

%

0

100

m/z750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950

%

0

100

m/z750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950

%

0

100

ASS_006_029_6 1024 (8.870) Sm (SG, 2x2.00); Cm (1008:1041-(989:1004+1043:1057)) 1: TOF MS ES+ 1.45e4974.81

968.82

838.82 968.09

928.95

928.17 968.01

1012.65

1012.731113.04

1013.68

1014.36

1062.43

1113.86

1173.361116.88

1123.801173.48

1174.58 1246.77

1238.11

1184.641249.84


967.69

967.63

967.56

927.39927.30

890.33838.85 927.68

1112.721059.71

1012.48

1013.27

1059.87

1112.55

1059.95

1061.24

1112.39

1066.03

1066.66

1171.09

1112.95

1170.99

1170.93

1113.671114.46

1115.62

1120.08

1171.26

1171.35

1236.23

1172.21

1172.38

1176.92

1236.34

1308.881237.23

1237.33

1308.57

1309.84

1390.59

1316.37 1483.141397.66

ASS_006_029_S 1050 (9.098) Sm (SG, 2x2.00); Cm (1014:1079) 1: TOF MS ES+ 4.78e51068.24

1019.78

974.82

838.83

934.84

897.48

854.81

935.54

936.22

976.14

976.84

977.48

978.22

1020.50

1021.27

1021.86

1068.34

1121.60

1069.81

1070.54

1071.19

1072.81

1122.45 1180.59

1123.25

1124.00

1124.70

1127.39

1128.76

1181.43

1182.271246.11

1183.16

1186.58

1247.07

1247.95

ASS-006-029-4 on ice 60min 1796-33 0.1%TFA BEH130 C18 #186003556 (0228341401)

m/z990 995 1000 1005 1010 1015 1020 1025 1030 1035 1040 1045

%

0

100

m/z990 995 1000 1005 1010 1015 1020 1025 1030 1035 1040 1045

%

0

100

m/z990 995 1000 1005 1010 1015 1020 1025 1030 1035 1040 1045

%

0

100


1011.89

1011.79

1011.74

990.781010.36

1005.071002.53991.14 1001.46

995.69 998.051008.58

1005.53

1012.73

1013.68

1014.17

1021.551016.33

1020.271017.23 1022.281025.27

1024.98

1027.06

1027.98

1030.13


1011.50

1010.22

1009.66998.33990.78996.76995.65

993.48

1001.86 1006.66

1004.81

1012.48

1013.27

1015.35

1024.141018.61 1019.76

1029.901025.251037.701033.041036.03 1043.19

1039.81

ASS_006_029_S 1050 (9.098) Sm (SG, 2x2.00); Cm (1014:1079) 1: TOF MS ES+ 4.37e51019.78

1019.00

990.79 1018.27

1017.511012.52

1002.23996.50991.79993.50

997.50

1000.23 1006.76 1009.93

1020.50

1021.27

1021.86

1022.64

1024.17

1025.15

1025.50

1026.55

1032.761028.521033.52

1034.52

Start

0 ℃

24 ℃

Calcd: [M+22H]22+ 1019.78

Found: [M+22H]22+ 1019.78

Calcd: [M+22H]22+ 1011.58

Found: [M+22H]22+ 1011.50

Start

0 ℃

24 ℃

Could determine the reaction conditions for glycoprotein ・Racemization of glycoprotein was also monitored by MS.

・Deprotection of glycoprotein should be carried out at 0 ℃.

Conclusion Hydrogen/Deuterium exchange (HDX)/MS has successfully revealed partial

racemization of amino acid residues during chemical synthesis of

glycopeptides and glycoproteins upon the removal of the sialic acid benzyl

groups under alkaline conditions. This system can be used to synthesize

glycopeptides/glycoproteins accurately.

J. Am. Chem. Soc. 2012, 134, 5428-5431

Calcd: [M+22H]22+ 1011.58

Found: broadening

Lyophilization (with H2O)

Twice

1.50 mM NaOD,

D2O, Temp.,60 min

2. Lyophilization

(with H2O), Twice

During the removal of the sialic acid benzyl groups under alkaline conditions, racemization

will occur.

Fully chemically-synthetic route

Chemical Synthesis of Interferon b-1a

180 min

Found: [M+3H] 3+ (1157.99)

1158.24

60 min

Found: [M+3H]3+ 1157.99

30 min

Found: [M+3H]3+ 1157.99

Spectrum:

Monitored the shift of the monoisotopic

mass with time.

Time (min)

m/z: [M+3]3+

Plot of monoisotopic’ centroid.

50 ℃:

Found: [M+3H]3+ 1160.56

24 ℃:

Found: [M+3H]3+ 1158.48

0 ℃:

Found: [M+3H]3+ 1157.99

Chromatogram (TIC): Spectrum: Calcd.:[M+3H]3+1157.98

Spectrum:

Monitored the shift of the monoisotopic

mass with temperature.

Temp.: 24 ℃

With this method, peaks will not

be visible if the reaction

conditions are unsuitable.

Deuterium attached via enolization of the carbonyl Cα-H were

monitored by this system.

simple and quick ms analysis of chemically synthesized … · 2017. 6. 16. · simple and quick ms...

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