釉原蛋白微损分析方法鉴定人骨性别的有效性与优化

doi:10.16359/j.1000-3193/AAS.2023.0018

人类学学报 ›› 2023, Vol. 42 ›› Issue (04): 472-487.doi: 10.16359/j.1000-3193/AAS.2023.0018

釉原蛋白微损分析方法鉴定人骨性别的有效性与优化

张宝帅¹^,²(), 吴晓桐¹^,², 吴高³, 金正耀¹^,², 范安川¹^,²()

1.中国科学技术大学科技考古实验室，合肥 230000
2.中国科学技术大学科技史与科技考古系，合肥 230000
3.中国科学技术大学生命科学实验中心，合肥 230000

收稿日期:2021-12-07 修回日期:2022-06-08 出版日期:2023-08-15 发布日期:2023-08-10
通讯作者: 范安川，副教授，主要从事科技考古研究。E-mail: anchuan@ustc.edu.cn
作者简介:张宝帅，硕士研究生，主要从事古蛋白质组学研究。E-mail: baoshuai@mail.ustc.edu.cn
基金资助:
国家自然科学基金(41303080);中国科学院青年创新促进会会员人才项目(2018499);中国科学技术大学学术领军人才培养计划(2018)

Verification and optimization of micro-destructive proteomics method for sex determination of archaeological human remains

ZHANG Baoshuai¹^,²(), WU Xiaotong¹^,², WU Gao³, JIN Zhengyao¹^,², FAN Anchuan¹^,²()

1. USTC Archaeometry Laboratory, University of Science and Technology of China, Hefei 230000
2. Department for the History of Science and Scientific Archaeology, University of Science and Technology of China, Hefei 230000
3. Core Facility Center for Life Sciences, University of Science and Technology of China, Hefei 230000

Received:2021-12-07 Revised:2022-06-08 Online:2023-08-15 Published:2023-08-10

摘要/Abstract

摘要：

釉原蛋白微损分析方法是一种新的考古人类遗骸性别鉴定方法。最新的研究表明，该方法可以对未成年个体和保存较差的人类遗骸进行可靠的性别鉴定，而中国尚未开展相关研究。为验证和优化基于微损取样的釉原蛋白性别鉴定方法，本文对中国不同地区新石器时代至秦汉时期11个遗址的24个样品进行了研究。结果表明，基于该方法测试的所有样本都获得了可靠的性别信息。同时，本文对该方法的实验步骤和性别判定标准进行了优化：1）根据所获43组实验结果，提出以30个肽段作为排除男性假阴性可能的参考阈值；2）对盐酸溶液中的多肽进行多次提取是增加多肽数目和种类的有效途径，同时不会对样品造成二次破坏；3）数据检索过程中，Oxidation(M)和Deamidation(NQ)三种可变修饰对获得可靠的性别鉴定结果是必要的。

关键词: 釉原蛋白, 性别鉴定

Abstract:

Proteomics analysis of sex-specific amelogenin peptides in tooth enamel has a high accuracy in determining sex, which is especially useful for poorly preserved fossil human remains. Destructive sampling has been used in most studies, which consumed a considerable been used in most studies, so less destructive analysis such as the one described here is more desirable. Since this method was developed, many researchers have used it to identify sex of archaeological samples. To verify and optimize this approach, the use of micro-destructive analysis of teeth with different preservation conditions is investigated using 24 samples from 11 sites (from Neolithic to the Qin-Han period). This method obtained sex identification results consistent with physical anthropology in all 18 archaeological samples with known sex. At the same time, six individuals of unknown sex were reliably judged, which shows that the micro-destructive analysis method has good applicability to tooth samples. We optimized this method in sampling and data analysis, noting the following points. 1) There is a certain linear relationship between number of total peptides and Ay-specific peptides. Combined with the fitting curve, 30 peptide fragments were identified as the reference threshold to exclude male false negative results. For samples without Ay-specific peptide segments, if the value was higher than 30, it was determined to be female; otherwise it’s sex was not determined. 2) Multiple extraction of peptides from HCl solution is an effective way to increase number and types of peptides without secondary damage to teeth. 3) Three kinds of variable modification (Oxidation (M) and Deamidation (NQ)) are necessary in database search. At the same time, the relationship between different factors and protein content was also evaluated. At present, there is no relationship between burial or thermal age, distribution area and preservation status of amelogenin peptides. How to characterize the preservation status of amelogenin in samples by appearance or other means remains to be further studied. The data obtained in this study also offers a research basis for further discussion of mechanisms affecting the preservation of amelogenin peptides. Therefore, experiments to evaluate preservation of enamel protein before proteomic analysis is an effective method to minimize the damage of samples.

Key words: Amelogenin, Sex identification

中图分类号:

Q987

张宝帅, 吴晓桐, 吴高, 金正耀, 范安川. 釉原蛋白微损分析方法鉴定人骨性别的有效性与优化[J]. 人类学学报, 2023, 42(04): 472-487.

ZHANG Baoshuai, WU Xiaotong, WU Gao, JIN Zhengyao, FAN Anchuan. Verification and optimization of micro-destructive proteomics method for sex determination of archaeological human remains[J]. Acta Anthropologica Sinica, 2023, 42(04): 472-487.

图/表 10

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序号No.	标本号 Sample	成年 adult	性别 sex	遗址名称site	遗址位置 location	年代age	热年龄 thermal age(BP)
1	M348	Y	M	杨官寨	陕西西安	YS²(3500-3000 BC)	47210
2	M491	Y	F	杨官寨	陕西西安	YS²(3500-3000 BC)	47210
3	M426	Y	F	杨官寨	陕西西安	YS²(3500-3000 BC)	47210
4	M158:2	Y	M	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
5	M99	Y	M	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
6	M111	Y	M	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
7	M50	Y	F	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
8	M374	Y	M	下靳	山西临汾	TS(2300-1900 BC)	26073
9	M447	Y	M	下靳	山西临汾	TS(2300-1900 BC)	26073
10	M83	Y	F	下靳	山西临汾	TS(2300-1900 BC)	26073
11	H393(1)	Y	M	城子崖	山东济南	YU(1900-1500 BC)	32227
12	M75	Y	M	丁公	山东滨州	LS(2400-2000 BC)	36529
13	M27	Y	M	丁公	山东滨州	LS(2400-2000 BC)	36529
14	SJCM62	?	?	苏家村	山东日照	LS(2400-2000 BC)	13331
15	2004 ZL M26	Y	?	银子坛	贵州威宁	ZH(400 BC-25 AD)	6514
16	M6:A	Y	F	纱帽山	云南昆明	ZH(250 BC-55 AD)	23188
17	T8M52	Y	M	纱帽山	云南昆明	ZH(250 BC-55 AD)	23188
18	M22-1 A	Y	M	纱帽山	云南昆明	ZH(250 BC-55 AD)	23188
19	M28A	Y	?	艾斯克霞尔南	新疆哈密	TI¹(700-400 BC)	65600
20	M28B	Y	?	艾斯克霞尔南	新疆哈密	TI¹(700-400 BC)	65600
21	SAY M258	Y	M	洋海	新疆吐鲁番	QH(公元前13世纪-前3世纪)	127228
22	SAY M280	Y	F	洋海	新疆吐鲁番	QH(公元前13世纪-前3世纪)	127228
23	M47	Y	?	辛店	河南安阳	SH³(1300-1000 BC)	33324
24	H340(3)	Y	?	辛店	河南安阳	SH³(1300-1000 BC)	33324

序号No.	标本号 Sample	成年 adult	性别 sex	遗址名称site	遗址位置 location	年代age	热年龄 thermal age(BP)
1	M348	Y	M	杨官寨	陕西西安	YS²(3500-3000 BC)	47210
2	M491	Y	F	杨官寨	陕西西安	YS²(3500-3000 BC)	47210
3	M426	Y	F	杨官寨	陕西西安	YS²(3500-3000 BC)	47210
4	M158:2	Y	M	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
5	M99	Y	M	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
6	M111	Y	M	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
7	M50	Y	F	蒋庄	江苏兴化	LZ(3300-2300 BC)	43215
8	M374	Y	M	下靳	山西临汾	TS(2300-1900 BC)	26073
9	M447	Y	M	下靳	山西临汾	TS(2300-1900 BC)	26073
10	M83	Y	F	下靳	山西临汾	TS(2300-1900 BC)	26073
11	H393(1)	Y	M	城子崖	山东济南	YU(1900-1500 BC)	32227
12	M75	Y	M	丁公	山东滨州	LS(2400-2000 BC)	36529
13	M27	Y	M	丁公	山东滨州	LS(2400-2000 BC)	36529
14	SJCM62	?	?	苏家村	山东日照	LS(2400-2000 BC)	13331
15	2004 ZL M26	Y	?	银子坛	贵州威宁	ZH(400 BC-25 AD)	6514
16	M6:A	Y	F	纱帽山	云南昆明	ZH(250 BC-55 AD)	23188
17	T8M52	Y	M	纱帽山	云南昆明	ZH(250 BC-55 AD)	23188
18	M22-1 A	Y	M	纱帽山	云南昆明	ZH(250 BC-55 AD)	23188
19	M28A	Y	?	艾斯克霞尔南	新疆哈密	TI¹(700-400 BC)	65600
20	M28B	Y	?	艾斯克霞尔南	新疆哈密	TI¹(700-400 BC)	65600
21	SAY M258	Y	M	洋海	新疆吐鲁番	QH(公元前13世纪-前3世纪)	127228
22	SAY M280	Y	F	洋海	新疆吐鲁番	QH(公元前13世纪-前3世纪)	127228
23	M47	Y	?	辛店	河南安阳	SH³(1300-1000 BC)	33324
24	H340(3)	Y	?	辛店	河南安阳	SH³(1300-1000 BC)	33324

编号No.	刻蚀时间 Etching time(min)	牙钻清洁 clean with drill	性别 Sex	方法method					Method III
				I	II	III	IV	V	Total peptides		Ay
				I	II	III	IV	V	肽段数	PSM	肽段数	PSM
1	30	no	M	M	M	M	M	M	74	140	4	5
2-1	30	no	F	F	F	F	F	F	178	508	N/A	N/A
2-2	15	no	F	F	F	F	F	F	145	501	N/A	N/A
3	30	no	F	F	F	F	F	F	182	487	N/A	N/A
4-1	10	yes	M	M	M	M	M	M	113	332	12	21
4-2	10	yes	M	M	M	M	M	M	120	371	19	28
4-3	10	yes	M	M	M	M	M	M	108	327	11	21
5	15	yes	M	F	M	M	M	M	31	44	2	3
6-1	10	yes	M	F	M	M	M	M	74	294	7	8
6-2	10	yes	M	M	M	M	M	M	79	251	7	14
6-3	10	yes	M	F	M	M	M	M	45	166	2	5
7	30	yes	F	F	F	F	F	F	56	158	N/A	N/A
8	2	yes	M	F	M	M	M	M	74	196	4	6
9	10	yes	M	M	M	M	M	M	201	656	17	31
10	2	yes	F	F	F	F	F	F	43	126	N/A	N/A
11-1	5	yes	M	F	M	M	M	M	79	216	10	15
11-2	5	yes	M	M	M	M	M	M	83	225	12	18
11-3	5	yes	M	M	M	M	M	M	158	512	25	41
12-1	10	yes	M	F	M	M	M	M	53	139	3	5
12-2	5	yes	M	F	M	M	M	M	56	152	4	4
12-3	5	yes	M	F	M	M	M	M	87	234	10	17
12-4	30	yes	M	F	M	M	M	M	72	204	7	9
13-1	5	yes	M	F	F	M	M	M	20	53	2	2
13-2	5	yes	M	F	F	M	M	M	18	34	2	2
14	30	yes	?	F	F	F	F	F	31	56	N/A	N/A
15	2	yes	?	F	F	F	F	F	48	118	N/A	N/A
16-1	5	no	F	F	F	F	F	F	8	11	N/A	N/A
16-2	5	yes	F	F	F	F	F	F	52	140	N/A	N/A
17-1	5	yes	M	F	M	M	M	M	44	97	4	6
17-2	5	yes	M	M	M	M	M	M	122	276	20	25
17-3	5	yes	M	F	M	M	M	M	83	208	11	18
18-1	10	yes	M	M	M	M	M	M	133	441	20	26
18-2	5	yes	M	M	M	M	M	M	112	365	13	29
18-3	5	yes	M	F	M	M	M	M	70	209	5	8
19	30	yes	?	F	F	F	F	F	65	181	N/A	N/A
20	30	yes	?	F	F	F	F	F	65	182	N/A	N/A
21-1	30	no	M	F	F	F	F	F	20	38	N/A	N/A
21-2	30	yes	M	F	M	M	M	M	77	284	2	5
22	30	yes	F	F	F	F	F	F	30	72	N/A	N/A
23	30	yes	?	M	M	M	M	M	68	248	7	10
24-1	15	yes	?	F	F	F	F	F	199	986	N/A	N/A
24-2	15	yes	?	F	F	F	F	F	178	889	N/A	N/A
24-3	15	yes	?	F	F	F	F	F	112	547	N/A	N/A

编号No.	刻蚀时间 Etching time(min)	牙钻清洁 clean with drill	性别 Sex	方法method					Method III
				I	II	III	IV	V	Total peptides		Ay
				I	II	III	IV	V	肽段数	PSM	肽段数	PSM
1	30	no	M	M	M	M	M	M	74	140	4	5
2-1	30	no	F	F	F	F	F	F	178	508	N/A	N/A
2-2	15	no	F	F	F	F	F	F	145	501	N/A	N/A
3	30	no	F	F	F	F	F	F	182	487	N/A	N/A
4-1	10	yes	M	M	M	M	M	M	113	332	12	21
4-2	10	yes	M	M	M	M	M	M	120	371	19	28
4-3	10	yes	M	M	M	M	M	M	108	327	11	21
5	15	yes	M	F	M	M	M	M	31	44	2	3
6-1	10	yes	M	F	M	M	M	M	74	294	7	8
6-2	10	yes	M	M	M	M	M	M	79	251	7	14
6-3	10	yes	M	F	M	M	M	M	45	166	2	5
7	30	yes	F	F	F	F	F	F	56	158	N/A	N/A
8	2	yes	M	F	M	M	M	M	74	196	4	6
9	10	yes	M	M	M	M	M	M	201	656	17	31
10	2	yes	F	F	F	F	F	F	43	126	N/A	N/A
11-1	5	yes	M	F	M	M	M	M	79	216	10	15
11-2	5	yes	M	M	M	M	M	M	83	225	12	18
11-3	5	yes	M	M	M	M	M	M	158	512	25	41
12-1	10	yes	M	F	M	M	M	M	53	139	3	5
12-2	5	yes	M	F	M	M	M	M	56	152	4	4
12-3	5	yes	M	F	M	M	M	M	87	234	10	17
12-4	30	yes	M	F	M	M	M	M	72	204	7	9
13-1	5	yes	M	F	F	M	M	M	20	53	2	2
13-2	5	yes	M	F	F	M	M	M	18	34	2	2
14	30	yes	?	F	F	F	F	F	31	56	N/A	N/A
15	2	yes	?	F	F	F	F	F	48	118	N/A	N/A
16-1	5	no	F	F	F	F	F	F	8	11	N/A	N/A
16-2	5	yes	F	F	F	F	F	F	52	140	N/A	N/A
17-1	5	yes	M	F	M	M	M	M	44	97	4	6
17-2	5	yes	M	M	M	M	M	M	122	276	20	25
17-3	5	yes	M	F	M	M	M	M	83	208	11	18
18-1	10	yes	M	M	M	M	M	M	133	441	20	26
18-2	5	yes	M	M	M	M	M	M	112	365	13	29
18-3	5	yes	M	F	M	M	M	M	70	209	5	8
19	30	yes	?	F	F	F	F	F	65	181	N/A	N/A
20	30	yes	?	F	F	F	F	F	65	182	N/A	N/A
21-1	30	no	M	F	F	F	F	F	20	38	N/A	N/A
21-2	30	yes	M	F	M	M	M	M	77	284	2	5
22	30	yes	F	F	F	F	F	F	30	72	N/A	N/A
23	30	yes	?	M	M	M	M	M	68	248	7	10
24-1	15	yes	?	F	F	F	F	F	199	986	N/A	N/A
24-2	15	yes	?	F	F	F	F	F	178	889	N/A	N/A
24-3	15	yes	?	F	F	F	F	F	112	547	N/A	N/A

釉原蛋白微损分析方法鉴定人骨性别的有效性与优化

Verification and optimization of micro-destructive proteomics method for sex determination of archaeological human remains

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[1]	杨稳, 刘晓宁, 刘雄乐, 朱丽品. 结合改进卷积神经网络和最小二乘法的颅骨性别鉴定[J]. 人类学学报, 2019, 38(02): 265-275.
[2]	赵凌霞. 新石器时代人骨遗骸中古代DNA的提取及X-Y染色体同源基因片段的PCR扩增[J]. 人类学学报, 1996, 15(03): 200-209.
[3]	孙尚辉,欧永章. 国人坐骨大切迹的测量与性别判别分析[J]. 人类学学报, 1986, 5(04): 368-371.