质谱法定量测定高密度脂蛋白结合蛋白的糖基化水平

doi:10.7503/cjcu20180782

摘要/Abstract

摘要：

采用质谱法对4种高密度脂蛋白(HDL)的结合蛋白重组人载脂蛋白血清淀粉样蛋白A(SAA)、 α1-抗胰蛋白酶(A1AT)、 α2-人体血清糖蛋白(A2HSG)和A载脂蛋白C3(Apo C3)从蛋白质含量(蛋白的绝对定量)、位点特异性糖基化(糖肽的相对定量)及聚糖位点占有率等方面进行了研究. 利用四极杆-飞行时间质谱仪(Q-TOF)测量糖蛋白标样酶解产物的二级质谱碎片离子, 用Byonic软件发现了新的糖基化位点信息, 即增加了原位点处聚糖糖型的种类. 对于A2HSG, 新增了N-糖基化156位点上的4种糖型, N-糖基化176位点上的6种糖型, O-糖基化319位点的4种O-聚糖和O-糖基化346位点上的1种糖型. 对于Apo C3, 只有O-糖基化94一个位点, 在此位点上新增了9种糖型. 同时, 调整了用于定量蛋白的多肽, 使得定量更加准确. 采用三重四极杆串级质谱仪(UPLC-ESI-QQQ)研究了4种结合蛋白中多肽和糖肽的多反应监测(MRM)行为, 并重新计算了每种聚糖的位点占有率, 优化了现有的定量方法.

关键词: 质谱法, 高密度脂蛋白的结合蛋白, 定点糖基化, 新糖型, 多反应监测

Abstract:

The protein content, site-specific glycosylation, and site occupancy of glycan and so on for serum amyloid A(SAA), α-1-antitrypsin(A1AT), α-2-HS-glycoprotein(A2HSG) and apolipoprotein C-Ⅲ(Apo C3) bound to high density lipoproteins(HDL) were investigated by mass spectrometry. Four kinds of trypsin digested protein standard samples were fragmented in liquid phase-quadrupole time-of-flight mass spectrometer. Some new glycosylation sites and new glycoforms were found by analyzing and matching fragment ions. For A2HSG, one O-glycosylation site and four glycoforms at that site were found, as well as 11 glycoforms at the original three sites. For Apo C3, 9 glycoforms at the original site were founded. Meanwhile, a unique peptide that used to quantify the protein were adjusted to make the quantification more accurate. Moreover, the multiple reaction monitor(MRM) behavior of peptides and glycopeptides in four proteins were studied and the site occupancy rate of each glycoform were recalculated. It is expected that our new findings in the process of exploration of site-specific glycosylation of HDL bound SAA, A1AT, A2HSG and Apo C3 will provide valuable informations in search of new HDL biomarks and will probably be practically applied in clinical detection of diseases related to HDL.

Key words: Mass spectrometry, Binding proteins to high density lipoprotein, Site-specific glycosylation, New glycoform, Multiple reaction monitoring

中图分类号:

O657.6

TrendMD:

魏王慧, 储艳秋, 陈鹰, 高艳秋, 丁传凡. 质谱法定量测定高密度脂蛋白结合蛋白的糖基化水平. 高等学校化学学报, 2019, 40(6): 1141.

WEI Wanghui,CHU Yanqiu,CHEN Ying,Gao Yanqiu,DING Chuanfan. Quantitative Determination of the Glycosylation Level for the Binding Proteins to High Density Lipoprotein by Mass Spectrometry^†. Chem. J. Chinese Universities, 2019, 40(6): 1141.

图/表 11

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No.	Protein	Peptidesequence	Site	Glycan moiety	MS(calcd.), m/z		Retention time/min
No.	Protein	Peptidesequence	Site	Glycan moiety	Precursor ion	Product ion	Retention time/min
1	SAA	GPGGVWAAEAISDAR			486.4	448.2, 561.3	6.82
2	A1AT	AVLTIDEK			444.8	718.4, 605.3	4.99
3	A1AT	QLAHQSNSTNIFFSPVSIATAFAMLSLGTK	70	5_4_0_2	1078.5	366.1	10.58
4	A1AT	QLAHQSNSTNIFFSPVSIATAFAMLSLGTK	70	5_4_1_2	1107.7	366.1	10.29
5	A1AT	ADTHDEILEGLNFNLTEIPEAQIHEGFQELLR	107	5_4_1_1	1151.6	366.1	9.15
6	A1AT	ADTHDEILEGLNFNLTEIPEAQIHEGFQELLR	107	6_5_0_3	1311.8	366.1	9.43
7	A1AT	ADTHDEILEGLNFNLTEIPEAQIHEGFQELLR	107	6_5_1_3	1341.0	366.1	9.43
8	A1AT	YLGNATAIFFLPDEGK	271	5_4_0_2	991.2	366.1	8.23
9	A1AT	YLGNATAIFFLPDEGK	271	5_4_1_2	1027.7	366.1	6.74

No.	Protein	Peptidesequence	Site	Glycan moiety	MS(calcd.), m/z		Retention time/min
No.	Protein	Peptidesequence	Site	Glycan moiety	Precursor ion	Product ion	Retention time/min
1	SAA	GPGGVWAAEAISDAR			486.4	448.2, 561.3	6.82
2	A1AT	AVLTIDEK			444.8	718.4, 605.3	4.99
3	A1AT	QLAHQSNSTNIFFSPVSIATAFAMLSLGTK	70	5_4_0_2	1078.5	366.1	10.58
4	A1AT	QLAHQSNSTNIFFSPVSIATAFAMLSLGTK	70	5_4_1_2	1107.7	366.1	10.29
5	A1AT	ADTHDEILEGLNFNLTEIPEAQIHEGFQELLR	107	5_4_1_1	1151.6	366.1	9.15
6	A1AT	ADTHDEILEGLNFNLTEIPEAQIHEGFQELLR	107	6_5_0_3	1311.8	366.1	9.43
7	A1AT	ADTHDEILEGLNFNLTEIPEAQIHEGFQELLR	107	6_5_1_3	1341.0	366.1	9.43
8	A1AT	YLGNATAIFFLPDEGK	271	5_4_0_2	991.2	366.1	8.23
9	A1AT	YLGNATAIFFLPDEGK	271	5_4_1_2	1027.7	366.1	6.74

No.	Protein	Peptidesequence	Site	Glycan moiety	MS(calcd.), m/z		Retention time/min
No.	Protein	Peptidesequence	Site	Glycan moiety	Precursor ion	Product ion	Retention time/min
1	A2HSG	EATEAAK			360.2	289.1, 519.3	2.84
2	A2HSG	VCQDCPLLAPLNDTR	156	5_4_0_1	1191.2	366.1	6.87
3	A2HSG	VCQDCPLLAPLNDTR	156	5_4_0_2	1288.2	366.1	7.03
4	A2HSG	VCQDCPLLAPLNDTR	156	5_4_1_2	1002.9	366.1	6.99
5	A2HSG	VCQDCPLLAPLNDTR	156	5_4_2_1	966.6	366.1	7.01
6	A2HSG	VCQDCPLLAPLNDTR	156	6_5_0_2	1057.7	366.1	6.99
7	A2HSG	VCQDCPLLAPLNDTR	156	6_5_0_3	1130.5	366.1	7.08
8	A2HSG	VCQDCPLLAPLNDTR	156	6_5_1_0	948.6	366.1	7.17
9	A2HSG	VCQDCPLLAPLNDTR	156	6_5_1_3	1167.0	366.1	7.06
10	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_0_1	1070.4	366.1	7.11
11	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_0_2	1143.0	366.1	7.22
12	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_1_2	1179.7	366.1	7.2
13	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_3_1	1180.3	366.1	7.2
14	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_0_1	1161.7	366.1	7.09
15	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_0_2	1234.3	366.1	7.17
16	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_0_3	1307.1	366.1	7.3
No.	Protein	Peptidesequence	Site	Glycan moiety	MS(calcd.), m/z		Retention time/min
No.	Protein	Peptidesequence	Site	Glycan moiety	Precursor ion	Product ion	Retention time/min
17	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_1_2	1271.0	366.1	7.17
18	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_1_3	1343.8	366.1	7.3
19	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	7_6_0_0	1180.5	366.1	7.2
20	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_1_0_1	913.1	274.1	11.02
21	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_1_0_2	757.8	274.1	7.89
22	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_1_1_1	961.8	274.1	11.06
23	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_2_0_1	735.8	274.1	2.69
24	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	1_1_0_1	891.4	274.1	6.32
25	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	1_1_0_2	988.5	274.1	6.34
26	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	2_1_1_0	897.1	366.1	11.08
27	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	2_2_0_0	916.1	366.1	7.21

No.	Protein	Peptidesequence	Site	Glycan moiety	MS(calcd.), m/z		Retention time/min
No.	Protein	Peptidesequence	Site	Glycan moiety	Precursor ion	Product ion	Retention time/min
1	A2HSG	EATEAAK			360.2	289.1, 519.3	2.84
2	A2HSG	VCQDCPLLAPLNDTR	156	5_4_0_1	1191.2	366.1	6.87
3	A2HSG	VCQDCPLLAPLNDTR	156	5_4_0_2	1288.2	366.1	7.03
4	A2HSG	VCQDCPLLAPLNDTR	156	5_4_1_2	1002.9	366.1	6.99
5	A2HSG	VCQDCPLLAPLNDTR	156	5_4_2_1	966.6	366.1	7.01
6	A2HSG	VCQDCPLLAPLNDTR	156	6_5_0_2	1057.7	366.1	6.99
7	A2HSG	VCQDCPLLAPLNDTR	156	6_5_0_3	1130.5	366.1	7.08
8	A2HSG	VCQDCPLLAPLNDTR	156	6_5_1_0	948.6	366.1	7.17
9	A2HSG	VCQDCPLLAPLNDTR	156	6_5_1_3	1167.0	366.1	7.06
10	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_0_1	1070.4	366.1	7.11
11	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_0_2	1143.0	366.1	7.22
12	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_1_2	1179.7	366.1	7.2
13	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	5_4_3_1	1180.3	366.1	7.2
14	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_0_1	1161.7	366.1	7.09
15	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_0_2	1234.3	366.1	7.17
16	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_0_3	1307.1	366.1	7.3
No.	Protein	Peptidesequence	Site	Glycan moiety	MS(calcd.), m/z		Retention time/min
No.	Protein	Peptidesequence	Site	Glycan moiety	Precursor ion	Product ion	Retention time/min
17	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_1_2	1271.0	366.1	7.17
18	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	6_5_1_3	1343.8	366.1	7.3
19	A2HSG	AALAAFNAQNNGSNFQLEEISR	176	7_6_0_0	1180.5	366.1	7.2
20	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_1_0_1	913.1	274.1	11.02
21	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_1_0_2	757.8	274.1	7.89
22	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_1_1_1	961.8	274.1	11.06
23	A2HSG	HTFMGVVSLGSPSGEVSHPR	319	1_2_0_1	735.8	274.1	2.69
24	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	1_1_0_1	891.4	274.1	6.32
25	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	1_1_0_2	988.5	274.1	6.34
26	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	2_1_1_0	897.1	366.1	11.08
27	A2HSG	TVVQPSVGAAAGPVVPPCPGR	346	2_2_0_0	916.1	366.1	7.21

No.	Protein	Peptidesequence	Site	Glycan moiety	MS(calcd.), m/z		Retention time/min
No.	Protein	Peptidesequence	Site	Glycan moiety	Precursor ion	Product ion	Retention time/min
1	Apo C3	GWVTDGFSSLK			598.8	244.2, 854.4	7.25
2	Apo C3	PTSAVAA	94	0_3_0_0	613.3	204.1	7.21
3	Apo C3	PTSAVAA	94	0_3_1_0	686.3	204.1	11.07
4	Apo C3	PTSAVAA	94	1_1_0_1	636.8	274.1	8.18
5	Apo C3	PTSAVAA	94	1_1_0_2	782.3	274.1	8.29
6	Apo C3	PTSAVAA	94	1_1_1_1	709.8	274.1	7.89
7	Apo C3	PTSAVAA	94	1_2_0_2	883.9	274.1	7.84
8	Apo C3	PTSAVAA	94	1_2_1_0	665.8	366.1	11.08
9	Apo C3	PTSAVAA	94	1_3_0_0	694.3	366.1	11.07
10	Apo C3	PTSAVAA	94	1_3_1_1	912.9	274.1	7.86
11	Apo C3	PTSAVAA	94	2_1_1_0	645.3	366.1	11.07
12	Apo C3	PTSAVAA	94	2_2_0_0	673.8	366.1	7.86
13	Apo C3	PTSAVAA	94	2_2_1_2	1037.9	274.1	6.32
14	Apo C3	PTSAVAA	94	2_2_2_0	673.8	274.1	7.03
15	Apo C3	PTSAVAA	94	2_2_3_0	892.9	366.1	10.58
16	Apo C3	PTSAVAA	94	2_3_0_1	920.9	274.1	7.60