古菌脂质的前处理方法比较及超高效液相色谱-高分辨质谱分析

doi:10.3724/SP.J.1123.2019.12009

色谱 ›› 2020, Vol. 38 ›› Issue (8): 914-922.DOI: 10.3724/SP.J.1123.2019.12009

古菌脂质的前处理方法比较及超高效液相色谱-高分辨质谱分析

王小雪¹^,², 何祉安¹^,², 李欣², 宋庆浩³, 邹欣薇²^,⁴, 宋雪瑶²^,⁴, 冯蕾¹^,²^,^*()

¹ 上海交通大学药学院, 上海 200240
² 上海交通大学分析测试中心, 上海 200240
³ 上海交通大学生命科学技术学院微生物代谢国家重点实验室, 上海 200240
⁴ 上海交通大学农业与生物学院, 上海 200240

收稿日期:2019-12-04 出版日期:2020-08-08 发布日期:2020-12-11
通讯作者: 冯蕾
作者简介:冯蕾.E-mail:fiona.fenglei@sjtu.edu.cn
基金资助:
国家自然科学基金青-科学基金项目(21106082);国家自然科学基金面上项目(41676121)

Comparison of pretreatment methods in lipid analysis and ultra-performance liquid chromatography-mass spectrometry analysis of archaea

WANG Xiaoxue¹^,², HE Zhi'an¹^,², LI Xin², SONG Qinghao³, ZOU Xinwei²^,⁴, SONG Xueyao²^,⁴, FENG Lei¹^,²^,^*()

¹ School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
² Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240, China
³ State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
⁴ School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2019-12-04 Online:2020-08-08 Published:2020-12-11
Contact: FENG Lei
Supported by:
Youth Program of National Natural Science Foundation of China(21106082);General Program of National Natural Science Foundation of China(41676121)

摘要/Abstract

摘要：

古菌是一类兼具真菌及细菌细胞特征、大多生活在极端环境下的单细胞微生物。由于膜结构及代谢通路的特殊性，研究古菌脂质对深入探究极端环境下生命的正常活动具有重要意义。前处理方法在组学分析中会极大影响最终结果的准确度及可靠性。该文基于超高效液相色谱-高分辨质谱联用（ultra-performance liquid chromatography-high resolution mass spectrometry，UPLC-HRMS）技术，以嗜热嗜压古菌Pyrococcus yayanosii为模式生物，采用Bligh-Dyer加酸法、Folch法、甲基叔丁基醚（methyl tert-butyl ether，MTBE）法及固相萃取（SPE）法提取脂质成分，从提取效率、重复性、歧视性等角度进行方法评价，并最终确定SPE法和MTBE法的提取重复性和提取效率最好，适合高通量古菌全脂质提取。利用SPE提取、高分辨质谱分析对P.yayanosii的常规脂质进行了全面分析，共鉴定到了1402种脂质。本研究旨在为古菌和其他极端微生物的非靶向脂质组学分析和脂质代谢研究提供方法参考。

关键词: 脂质组学, 提取, 质谱, 裂解规律, 嗜热嗜压菌, 古菌

Abstract:

Archaea are single-cell microorganisms, structurally and biochemically similar to bacteria and fungi. Most of them live in extreme environments, such as high salt, extremely acidic, extremely hot, and anaerobicenvironments. The membrane structure and related metabolic pathways of archaea are different from those of other microorganisms. Therefore, studying the lipid metabolism of archaea is of great significance for exploring the life activities in extreme environments. As the first step in lipidomic analysis, lipid extraction and pretreatment methods play an important role, as they influence the accuracy and reliability of the final results. We harnessed ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) to detect the total normal lipids. The hyperthermophilic archaeon Pyrococcus yayanosii was selected as the model. The Bligh-Dyer acidic method, Folch method, methyl tert-butyl ether (MTBE) method, and solid-phase extraction (SPE) method were compared by multi-component analysis in terms of extraction efficiency, reproducibility, and extraction discrimination. Comprehensive analysis revealed that the SPE and MTBE methods showed the best extraction repeatability and extraction efficiency, and were suitable for high-throughput microbial lipid extraction. Finally, normal lipid components of P. yayanosii were comprehensively analyzed by SPE coupled with UPLC-HRMS. A total of 1402 lipid components were identified. This article aims to provide a reference for non-targeted lipidomic analysis of archaea and other microorganisms towards understanding their lipid metabolism.

Key words: lipidomics, extraction, mass spectrometry (MS), fragmentation pathway, hyperthermophiles, archaea

王小雪, 何祉安, 李欣, 宋庆浩, 邹欣薇, 宋雪瑶, 冯蕾. 古菌脂质的前处理方法比较及超高效液相色谱-高分辨质谱分析[J]. 色谱, 2020, 38(8): 914-922.

WANG Xiaoxue, HE Zhi'an, LI Xin, SONG Qinghao, ZOU Xinwei, SONG Xueyao, FENG Lei. Comparison of pretreatment methods in lipid analysis and ultra-performance liquid chromatography-mass spectrometry analysis of archaea[J]. Chinese Journal of Chromatography, 2020, 38(8): 914-922.

图/表 13

表1 已报道的微生物脂质提取方法

Table 1 Reported lipid extraction methods for microorganisms

Extraction	Sample	Extracted lipid	Reference
GDGT: glycerol dialkyl glycerol tetraethers; MTBE: methyl tert-butyl ether; SPE: solid phase extraction.
Bligh-Dyer method	archaea, bacteria	GDGT	[25]
Folch method	Escherichia coli	Membrane lipid	[26]
MTBE method	microalgae	Fatty acid	[27, 28]
SPE method	bacteria	lipid-linked	[29]
		oligosaccharide

表2 Lipidsearch数据库物质匹配参数设置

Table 2 Parameters for library search by Lipidsearch software

Parameter	Setting value
Rt: retention time; ppm: 10^-6.
Search type	Product QEX
Precursor tol	5.0 ppm
Product tol	5.0 ppm
Rt range	±0.5 min
Adducts	+H, +NH₄, +Na, +2H

图1 TG(18:1/16:0/16:0) [M+NH4]+离子的二级质谱图及裂解途径

Fig. 1 MS/MS spectrum of TG(18:1/16:0/16:0) [M+NH4]+ ion and fragmentation pathway TG: triglyceride.

表3 部分TG和DG的二级质谱结果

Table 3 Major MS/MS results of triglycerides (TGs) and diglycerides (DGs)

Compound name	Formula	Adduct	m/z	Mass error/ppm	Fragments (m/z)
TG(15:0/14:0/16:0)	C₄₈H₉₂O₆	[M+NH₄]⁺	782.7220	-0.92	509.4564, 523.4719, 537.4870
		[M+Na]⁺	787.6772	-1.761
TG(15:0/16:1/17:1)	C₅₁H₉₄O₆	[M+NH₄]⁺	820.7387	-1.136	561.4870, 549.4878
TG(16:1/14:0/16:1)	C₄₇H₈₈O₆	[M+NH₄]⁺	792.70681	-0.953	547.4711, 521.4559
		[M+Na]⁺		-0.877
DG(18:0/16:0)	C₃₇H₇₂O₅	[M+Na]⁺	619.5254	-2.745	341.3045, 313.2734
DG(18:0/18:0)	C₃₉H₇₆O₅	[M+NH₄]⁺	642.6023	-1.143	607.5656, 341.3050

图2 Cer(d16:0/23:0) [M+H]+离子的二级质谱图及裂解途径

Fig. 2 MS/MS spectrum of Cer(d16:0/23:0) [M+H]+ ion and fragmentation pathway Cer: ceramide.

表4 部分鞘脂类物质二级质谱结果

Table 4 Major MS/MS results of sphingolipids

Compound name	Formula	Adduct	m/z	Mass error/ppm	Fragments (m/z)
SM: sphingomyelin; SPH: sphingosine; HexCer: hexose-ceramide.
Cer(d16:0/22:0)	C₃₈H₇₇O₃N	[M+H]⁺	596.5974	-0.302	578.5863, 256.2632, 340.3568
Cer(d18:0/24:0)	C₄₂H₈₅O₃N	[M+H]⁺	652.6596	-0.9	634.65, 368.3883, 284.295
SM(t18:1/16:0)	C₃₉H₇₉O₇N₂P	[M+H]⁺	719.5697	-0.073	687.526, 335.2565, 313.2734
SPH(t16:0)	C₁₆H₃₅O₃N	[M+H]⁺	290.2684	-1.748	272.2578, 242.2475
HexCer(d18:1/22:0)	C₄₆H₈₉O₈N	[M+H]⁺	784.6650	-1.363	604.6002, 264.2681, 236.2369
HexCer(d18:1/23:0)	C₅₃H₁₀₁O₁₃N	[M+H]⁺	960.7308	-3.899	311.2038, 264.268

图3 PC(16:0/13:0) [M+H]+离子的二级质谱图及裂解途径

Fig. 3 MS/MS spectrum of PC(16:0/13:0) [M+H]+ ion and fragmentation pathway PC: phosphatidylcholine.

表5 部分磷脂类物质的二级质谱结果

Table 5 Major MS/MS results of glycerophospholipids

Compound name	Formula	Adduct	m/z	Mass error/ppm	Fragments (m/z)
PE: phosphatidyl ethanolamine; PMe: phosphatidyl methanol; LPC: lysophosphatidylcholine; PG: phosphatidylglycerol.
PC(16:0/13:0)	C₃₇H₇₄O₈NP	[M+H]⁺	692.5277	4.520	557.4445, 314.2875, 282.2791
LPC(16:0)	C₂₄H₅₀O₇NP	[M+H]⁺	496.3399	0.458	184.0733, 104.1069
PE(16:0/17:0)	C₃₈H₇₅O₈P	[M+H]⁺	691.5288	2.265	453.2993, 435.2885, 379.2628
PMe(16:0/20:3)	C₄₀H₇₃O₈P	[M+H]⁺	713.5106	-1.346	475.2817, 457.2708
PG(18:1/18:1)	C₄₂H₇₉O₁₀P	[M+NH₄]⁺	792.5735	-1.724	603.5333, 283.2629, 311.2941

图4 不同提取方法获得的脂类物质的数目

Fig. 4 Numbers of lipids extracted by different methods BD: Bligh-Dyer acidic method.The vertical axis represents the number of identified substances in different peak response intervals. Black color represents the detected response interval above 500000. Dark gray represents the detected response interval from 10000 to 500000. Light gray represents the detected response interval less than 10000.

图5 不同提取方法的提取重复性分析

Fig. 5 The relative standard deviation of lipid metabolites by different pretreatments The vertical axis represents the number of identified substances in different intervals. Black color represents coefficient of variation (CV, n=3) is less than 10% among this group. Light gray color represents CV (n=3) between 10% and 20%. Dark gray color represents CV (n=3) between 20% and 30%. * indicates a significant difference (P < 0.05).

图6 不同提取方法提取(a)脂肪酸、(b)甘油酯、(c)甘油磷脂、(d)鞘脂的热图

Fig. 6 Heatmap of (a) fatty acids, (b) glycerollipids, (c) phosphoglycerollipids, (d) sphingolipids with different extraction methods

图7 P. yayanosii古菌样品中脂质成分的UPLC-HRMS总离子流基峰色谱图

Fig. 7 UPLC-HRMS base peak ion chromatogram of lipid substances extracted from P. yayanosii by SPE UPLC-HRMS: ultra-performance liquid chromatography coupled with high-resolution mass spectrometry.

图8 P. yayanosii脂质物质的组成

Fig. 8 Lipid components of P. yayanosii Percentage equals current type of identified substances divided by the total number of identified substances.

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古菌脂质的前处理方法比较及超高效液相色谱-高分辨质谱分析

Comparison of pretreatment methods in lipid analysis and ultra-performance liquid chromatography-mass spectrometry analysis of archaea

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