基于UHPLC-QTOF/MS技术RAW264.7巨噬细胞甘油磷脂成分的快速表征

引用本文

SHE Yu-qi, XIAO Xue-rong, WU Xia, FENG Yi-fan. Rapid analysis of glycerophospholipids in RAW264.7 macrophage with UHPLC-QTOF/MS[J]. Acta Pharm Sin, 2016, 51(9): 1451-1457. 复制到剪切板

佘玉奇, 肖雪蓉, 吴霞, 冯毅凡. 基于UHPLC-QTOF/MS技术RAW264.7巨噬细胞甘油磷脂成分的快速表征[J]. 药学学报, 2016, 51(9): 1451-1457. 复制到剪切板

基于UHPLC-QTOF/MS技术RAW264.7巨噬细胞甘油磷脂成分的快速表征

佘玉奇, 肖雪蓉, 吴霞, 冯毅凡

广东药科大学中心实验室, 广东广州 510006

收稿日期: 2016-03-30; 修回日期: 2016-04-28

基金项目: 国家自然科学基金资助项目（21275036，81202429）。

^*通讯作者: Tel: 86-20-39352522, Fax: 86-20-39252523,E-mail: wxiaxia@163.com;
E-mail: tfyfeng@aliyun.com

摘要: 采用超高效液相色谱-四级杆飞行时间高分辨质谱联用技术（UHPLC-QTOF/MS）对RA264.7巨噬细胞甘油磷脂成分进行结构推断，运用修饰后的Bligh-Dyer法提取甘油磷脂，以流动相A : 水（含10mmol·L^-1乙酸铵和0.25%乙酸），流动相B : 乙腈-异丙醇（1∶1）（含10mmol·L^-1乙酸铵和0.25% 乙酸）进行梯度洗脱，在ESI正负离子模式下进行数据采集。本实验推断出82个甘油磷脂成分，其中包括57个磷脂酰胆碱（PCs）、21个磷脂酰乙醇胺（PEs）、3个磷脂酰甘油（PGs）和1个磷脂酰肌醇（PI）。本文建立的基于UHPLC-QTOF/MS液质联用技术的RAW264.7巨噬细胞甘油磷脂类成分分析方法快速、简便、可靠。

关键词: RAW264.7巨噬细胞甘油磷脂磷脂酰胆碱磷脂酰乙醇胺磷脂酰甘油磷脂酰肌醇

Rapid analysis of glycerophospholipids in RAW264.7 macrophage with UHPLC-QTOF/MS

SHE Yu-qi, XIAO Xue-rong, WU Xia, FENG Yi-fan

Central Laboratory, Guangdong Pharmaceutical University, Guangzhou 510006, China

Abstract: An ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometric method was developed for rapid analysis of glycerophospholipids in RAW264.7 macrophage. The modified Bligh-Dyer was applied to extract glycerophospholipids from RAW264.7 macrophage. The target compounds, detected by mass spectrometry in ESI⁺ and ESI^- mode, were separated by gradient elution with mobile phase (A) water (containing 10mmol·L^-1 ammonium acetate and 0.25% acetic acid) and (B) acetonitrile/isopropanol (1∶1) (containing 10mmol·L^-1 ammonium acetate and 0.25% acetic acid). A total of 82 glycerophospholipids including 57 phosphatidylcholines (PCs), 21 phosphatidylethanolamines (PEs), three phosphatidylglycerols (PGs) and one phosphatidylinositol (PI) were deduced. The UHPLC-QTOF/MS method is rapid, simple and credible for targeting analysis of glycerophospholipids of RAW264.7 macrophage.

Key words: RAW264.7 macrophage glycerophospholipid phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol phosphatidylinositol

脂质不仅是细胞的保护屏障,也是细胞内信号分子的前体物质,调节膜运输,并且参与细胞信号转导^{[1, 2]}。脂质的代谢产物,如花生四烯酸类成分,参与炎症的发生,有报道认为炎症反应与脂类代谢紊乱息息相关^{[3, 4]}。细胞膜甘油磷脂代谢紊乱会导致各种炎症性疾病的发生^[5],如动脉粥样硬化^[6]、2型糖尿病^[7]、阿尔茨海默症^[8]和癌症^[9]等。

甘油磷脂是细胞膜脂质的主要组成成分,是细胞膜不可缺少的组成构架,作为调节和控制细胞功能与疾病的重要参与者。甘油磷脂的结构由甘油骨架、两条脂肪酸链 (sn-1、sn-2) 和磷酸头部组成^[10]。在哺乳动物细胞中,磷脂主要包括磷脂酸 (PA)、磷脂酰胆碱 (PC)、磷脂酰乙醇胺 (PE)、磷脂酰肌醇 (PI)、甘油磷脂酰甘油 (PG) 以及磷脂酰丝氨酸 (PS)。由于不同的磷脂酸头部、脂肪酸链及其含有的不饱和键数目,使得甘油磷脂的种类繁多,这就为磷脂成分的鉴定增添了不少难度。

电喷雾质谱 (ESI-MS),可在短时间内对不同种类的脂质成分进行快速分析,这为脂质的整体分析鉴定提供了核心技术支持^[11]。色谱质谱技术的联合使用极大地推动磷脂组学的发展,尤其是软电离离子化和高分辨质谱分析技术在脂质组学的分析应用。ESI-MS具有高分辨率、高灵敏度、高通量、样品前处理过程简单、可实现大批量自动化进样的特点,适合对磷脂混合物的定性定量研究^{[12, 13]}。1997年,Brügger等^[14]第一次利用ESI/MS技术对从仓鼠卵巢细胞中提取的几种磷脂进行分析, 包括PC、PE、PG、PI和PA。Lorenzen等^[15]运用UHPLC-ESI-MS对黄色黏球菌 (Myxococcus xanthus) 的脂质成分进行了全面系统的分析。Wu等^[16]通过UHPLC-QTOF/MS技术寻找非甾体抗炎药物干预RAW264细胞炎症状态下甘油磷脂的动态变化。Cífková等^[17]结合HILIC- HPLC-MS和多变量数据分析方法对人类乳腺癌周围的正常组织的脂质进行分析。Zeng等^[18]运用UHPLC- QTOF/MS分析T2DM模型大鼠的血浆磷脂代谢,寻找2型糖尿病生物标记物。Koivusalo等^[19]运用ESI-MS对磷脂定量分析发现磷脂的酰基链长度、不饱和度以及进样浓度和仪器的响应有密切的关系。

本实验采用液质联用技术对RAW264.7细胞甘油磷脂成分进行分析,旨在建立一种基于液质联用技术的快速、简便、可靠的RAW264.7巨噬细胞甘油磷脂类成分表征方法。

材料与方法

实验仪器及试剂 超高效液相色谱/四级杆飞行时间串联质谱仪 (Acquity UHPLC-QTOF/MS,美国Waters公司); BEH C₁₈柱 (2.1 mm× 50 mm,1.7 μm,Waters); Masslynx 4.1数据处理系统 (Waters); HHS型电热恒温水浴锅 (上海博迅实业有限公司); KDC- 220HR高速冷冻离心机 (科大创新股份有限公司); TDL80-2B低速离心机 (上海安亭科学仪器厂); HF90 CO₂培养箱 (上海力申科学仪器有限公司); 显微镜 (江南永新光学有限公司); SW-CJ-1FD超净台 (苏净安泰); WH-2微型涡旋混合仪 (上海沪西分析仪器厂)。

异丙醇 (色谱纯,德国Merck公司); 甲醇、乙腈 (色谱纯,上海安谱实验科技股份有限公司); 乙酸 (色谱纯,美国Aladdin公司); 亮氨酸脑啡肽 (美国Sigma公司); 纯水 (屈臣氏); RAW264.7巨噬细胞 (购自中国科学院细胞库); 胎牛血清 (FBS,Gibco); 高糖培养基 (DMEM,Gibco); 0.25% 胰蛋白酶 (Gibco); 双抗(北京康贝源科技有限责任公司); 磷酸盐缓冲溶液 (PBS,美国Hyclone公司); DMSO (美国Sigma公司); 甘油磷脂对照品: 1-棕榈酰基-2-油酰基-sn-丙三氧基-3-磷脂胆碱 > 99% {PC (16∶0/18∶1); [M+H]⁺ = 760}、1-棕榈酰基-2-羟基-sn-丙三氧基-3-磷脂胆碱>99% {PC (16∶0/0∶0); [M+H]⁺ = 496}、1-硬脂酰基- 2-羟基-sn-丙三氧基-3-磷脂乙醇胺 > 99% {PE (18∶0/ 0∶0); [M+H]⁺ = 482}、1-油酰基-2-羟基-sn-丙三氧基- 3-磷脂胆碱 > 99% {PC (18∶1/0∶0); [M+H]⁺ = 522} (Avanti polar lipids)。

细胞培养 细胞复苏后接种于培养皿中,培养基为DMEM∶FBS∶双抗=89∶10∶1,置于37 ℃、5% CO₂以及饱和湿度的孵育箱中培养,每两天更换培养基,以1∶3的比例进行传代。

甘油磷脂对照品的配制 精密称定各甘油磷脂对照品,用甲醇溶解配制成10 μg·mL⁻¹的对照品溶液。

甘油磷脂的提取 采用修饰后的Bligh-Dyer^{[20, 21]}法提取甘油磷脂。磷脂的前处理过程在低温下 (4 ℃) 操作。提取溶剂采用甲醇−0.1 mol·L⁻¹盐酸−氯仿 (1∶1∶1),涡旋静置分层时,保留有机相,弃去水相以除去非脂成分。具体提取过程如下: ① 将细胞 (100 mm培养皿) 置于冰袋上操作; ② 吸取冷1×PBS 5 mL清洗贴壁细胞两次; ③ 加入冷1×PBS 1 mL,将贴壁细胞完全刮落,吹匀,转移至离心管中; ④ 600×g,4 ℃,离心5 min,弃去上清液; ⑤ 迅速置于液氮中使其结冰粒,即可进行后续操作; ⑥ 加入冷的0.1 mol·L⁻¹盐酸−甲醇(1∶1,各400 μL),再加入氯仿400 μL; ⑦ 涡旋1 min,16 000×g、4 ℃离心8 min; ⑧ 取下层有机相,氮气吹干; ⑨ 加入甲醇−氯仿 (9∶1) 80 μL,复溶,转移至微量内衬管进行UHPLC-QTOF/MS分析。

甘油磷脂的UHPLC-QTOF/MS分析 色谱柱为Acquity UHPLC BEH C₁₈ (2.1 mm× 50 mm,1.7 μm)。流动相A: 水 (含10 mmol·L⁻¹乙酸铵和0.25% 乙酸),流动相B: 乙腈 (含10 mmol·L⁻¹乙酸铵和0.25% 乙酸)。梯度洗脱程序0～4 min,61%～81.4% B; 4～ 20 min,81.4%～90% B; 20～21 min,90%～100% B; 21～24 min,100% B。流速: 0.3 mL·min⁻¹; 进样量: 5 μL; 柱温: 50 ℃; 梯度洗脱曲线 = 7。

质谱条件: 采用浓度为500 ng·mL⁻¹亮氨酸−脑啡肽进行实时校正质量数。离子源为ESI源,分别在正负离子模式下进行质谱信号采集,亮氨酸−脑啡肽在ESI⁺、ESI⁻ 模式下的m/z分别为 [M+H]⁺ = 556.277 1,[M−H]⁻ = 554.261 5。质量数扫描离子范围设定为450～950 Da。

质谱参数设定如下: 毛细管电压: 3.0 kV,锥孔电压: 30 V,提取锥孔电压: 1.0 V,脱溶剂温度: 300 ℃,离子源温度: 100 ℃,离子能量: 1.0 V,碰撞能量: 6.0 V,反向锥孔气流: 50 L·h⁻¹,脱溶剂气流: 500 L·h⁻¹。

结果与讨论 1 RAW264.7巨噬细胞甘油磷脂成分的UHPLC- QTOF/MS分析鉴定

在正、负离子模式下,得到RAW264.7细胞甘油磷脂的总离子流图,如图 1所示。对总离子流图中每个峰进行推断分析,每个色谱峰含有多个甘油磷脂成分的质谱信息,保留时间相近,但能够通过质谱系统自主地选择目标离子进行MS/MS分析。根据化合物的精确分子量及其元素组成推断其分子式,限定误差范围为5 ppm以内,并结合特征性碎片,通过Lipid Maps磷脂数据库进行检索比对,推断得到RAW264.7巨噬细胞甘油磷脂成分 (表 1)。

Figure 1 Total-ion-current chromatograms of glycerophos;pholipids in RAW264.7 cells (A: ESI⁺,B: ESI⁻)

Table 1 Identification of glycerophospholipids in RAW264.7 macrophage

No.	t_R/min	Adduct ion	MS (m/z)	ppm	Formula	MS/MS (m/z)	Metabolite
1	1.37	[M+H]⁺	468.310 5	3.2	C₂₂H₄₇NO₇P	468,184	PC (0∶0/14∶0)
2	1.47	[M+H]⁺	500.276 3	−2.8	C₂₅H₄₃NO₇P	500,359,141	PE (20∶5/0∶0)
3	1.52	[M+H]⁺	468.308 5	−1.1	C₂₂H₄₇NO₇P	468,450,285,184	PC (14∶0/0∶0)
4	1.56	[M+H]⁺	494.324 9	0.4	C₂₄H₄₉NO₇P	494,311,184	PC (16∶1/0∶0)
		[M+CH₃COO]⁻	552.328 6	−2.7	C₂₆H₅₁NO₉P	478,253,168
5	1.75	[M+H]⁺	526.295 7	4.4	C₂₇H₄₅NO₇P	526,141	PE (22∶6/0∶0)
6	1.85	[M+H]⁺	502.293 7	0.6	C₂₅H₄₅NO₇P	502,361,141	PE (20∶4/0∶0)
		[M−H]⁻	500.278 3	1.2	C₂₅H₄₃NO₇P	500,303,196,140
7	1.89	[M−H]⁻	450.261 2	−2.0	C₂₁H₄₁NO₇P	450,253,196,140	PE (16∶1/0∶0)
							Continued
No.	t_R/min	Adduct ion	MS (m/z)	ppm	Formula	MS/MS (m/z)	Metabolite
8	2.22	[M+H]⁺	528.309 4	0.8	C₂₇H₄₇NO₇P	528,141	LysoPE (22∶5/0∶0)
9	2.21	[M+H]⁺	496.340 8	1.0	C₂₄H₅₁NO₇P	313,184	PC (0∶0/16∶0)
		[M+CH₃COO]⁻	554.346 1	0.5	C₂₅H₅₃NO₉P	554,480,255,168
10	2.27	[M+H]⁺	508.341 0	1.4	C₂₅H₅₁NO₇P	508,184	PC (17∶1/0∶0)
		[M+CH₃COO]⁻	566.343 8	−3.5	C₂₇H₅₃NO₉P	566,267,168
11	2.42	[M+H]⁺	504.317 1	−0.6	C₂₅H₄₇NO₇P	363,141	PE (20∶3/0∶0) or PE (0∶0/20∶3)
		[M−H]⁻	502.294 4	2.0	C₂₅H₄₅NO₇P	502,305,196,140
12	2.43	[M+H]⁺	482.325 7	2.1	C₂₃H₄₉NO₇P	482,341	LysoPE (0∶0/18∶0)
		[M+CH₃COO]⁻	540.330 7	1.1	C₂₅H₅₁NO₉P	540,196,153,140
13	2.44	[M+H]⁺	496.340 2	−0.2	C₂₄H₅₁NO₇P	496,478,313,184,104	PC (16∶0/0∶0)
		[M+CH₃COO]⁻	554.343 9	−3.4	C₂₅H₅₃NO₉P	480,255,224
14	2.44	[M+H]⁺	522.355 8	−0.2	C₂₆H₅₃NO₇P	522,504,445,339,184	PC (0∶0/18∶1)
		[M+CH₃COO]⁻	580.359 7	−2.9	C₂₈H₅₅NO₉P	580,506,281,168
15	2.69	[M+H]⁺	544.339 0	−1.1	C₂₈H₅₁NO₇P	544,361,104,86	PC (20∶4/0∶0)
16	2.80	[M+H]⁺	510.354 4	−3.1	C₂₅H₅₃NO₇P	510,141	PE (20∶0/0∶0)
17	2.80	[M+H]⁺	480.310 2	2.5	C₂₃H₄₇NO₇P	480,339,141	PE (18∶1/0∶0)
18	2.81	[M+H]⁺	522.357 4	2.7	C₂₆H₅₃NO₇P	522,184	PC (18∶1/0∶0)
		[M+CH₃COO]⁻	580.360 9	−0.9	C₂₈H₅₅NO₉P	580,506,281,168
19	2.90	[M+H]⁺	482.364 0	6.0	C₂₄H₅₃NO₆P	482,184	PC (O-16∶0/0∶0)
20	3.13	[M+H]⁺	508.376 2	−1.0	C₂₆H₅₅NO₆P	508,184	PC (P-18∶0/0∶0)
21	3.50	[M+H]⁺	532.339 8	1.7	C₂₇H₅₁NO₇P	532,514,184,104	PC (19∶3/0∶0)
22	3.61	[M+H]⁺	524.371 3	−0.6	C₂₆H₅₅NO₇P	524,506,447,341,184	PC (18∶0/0∶0)
		[M+CH₃COO]⁻	582.377 7	1.0	C₂₈H₅₇NO₉P	582,508,283,168
23	3.62	[M+H]⁺	546.355 1	−1.6	C₂₈H₅₃NO₇P	546,184	PC (20∶3/0∶0) or LysoPC (20∶3)
24	3.62	[M+H]⁺	550.388 3	1.8	C₂₈H₅₇NO₇P	550,184	PC (20∶1/0∶0)
25	3.70	[M+H]⁺	482.324 6	−0.2	C₂₃H₄₉NO₇P	482,341	PE (18∶0/0∶0)
		[M−H]⁻	480.308 0	−2.1	C₂₃H₄₇NO₇P	283,196,153,140
26	3.87	[M+H]⁺	508.344 4	8.1	C₂₅H₅₁NO₇P	508,367,141	PE (20∶1/0∶0)
		[M−H]⁻	506.323 9	−1.6	C₂₅H₄₉NO₇P	508,309,196,140
27	4.13	[M+H]⁺	510.391 7	−1.4	C₂₆H₅₇NO₆P	510,184	PC (O-16∶0/2∶0) or PC (O-18∶0/0∶0)
28	4.70	[M+H]⁺	536.378 2	−0.7	C₂₇H₅₅NO₇P	536,518,395	PE (22∶1/0∶0)
29	5.50	[M−H]⁻	658.480 7	−0.8	C₃₆H₆₉NO₇P	658,140	PE (P-16∶0/15∶1)
30	5.84	[M−H]⁻	819.520 1	3.1	C₄₆H₇₆O₁₀P	819,327,281,153	PG (22∶6/18∶1)
31	6.59	[M+H]⁺	678.507 7	0.4	C₃₆H₇₃NO₈P	678,495,184	PC (16∶0/12∶0)
		[M+CH₃COO]⁻	736.510 9	−2.7	C₃₈H₇₅NO₁₀P	736,662,255,199
32	6.59	[M+H]⁺	704.523 6	0.9	C₃₈H₇₅NO₈P	704,184	PC (14∶0/16∶1)
		[M+CH₃COO]⁻	762.526 9	−2.1	C₄₀H₇₇NO₁₀P	762,688,253,168
33	6.59	[M−H]⁻	833.515 1	−3.5	C₄₃H₇₈O₁₃P	579,551,281,253,297,241, 223,153	PI (16∶1/18∶1)
34	6.75	[M+H]⁺	730.537 4	−1.8	C₄₀H₇₇NO₈P	730,184	PC (16∶1/16∶1)
35	7.42	[M−H]⁻	773.535 5	2.8	C₄₂H₇₈O₁₀P	773,417,281,227,153	PG (18∶1/18∶1)
36	7.85	[M+H]⁺	732.551 0	−4.5	C₄₀H₇₉NO₈P	732,184	PC (14∶0/18∶1)
		[M+CH₃COO]⁻	790.556 6	−4.0	C₄₂H₈₁NO₁₀P	790,716,227,168
37	8.01	[M+H]⁺	754.538 3	0.7	C₄₂H₇₇NO₈P	755,571,695,86	PC (14∶0/20∶4)
38	8.12	[M+H]⁺	706.541 2	3.5	C₃₈H₇₇NO₈P	706,184	PC (14∶0/16∶0)
		[M+CH₃COO]⁻	764.542 4	−2.4	C₄₀H₇₉NO₁₀P	764,255,168
39	8.20	[M+H]⁺	782.571 9	1.7	C₄₄H₈₁NO₈P	783,723,599,184	PC (16∶0/20∶4)
40	8.22	[M−H]⁻	747.519 4	2.4	C₄₀H₇₆O₁₀P	747,465,391,281,255,171,153	PG (16∶0/18∶1)
41	8.31	[M+H]⁺	780.553 0	1.8	C₄₄H₇₉NO₈P	781,721,598	PC (16∶1/20∶4)
							Continued
No.	t_R/min	Adduct ion	MS (m/z)	ppm	Formula	MS/MS (m/z)	Metabolite
42	8.70	[M+H]⁺	758.570 4	0.5	C₄₂H₈₁NO₈P	758,184	PC (18∶1/16∶1)
		[M+CH₃COO]⁻	816.577 9	2.9	C₄₄H₈₃NO₁₀P	816,742,281,253
43	8.70	[M+H]⁺	784.586 3	0.9	C₄₄H₈₃NO₈P	784,184	PC (36∶3)
44	8.98	[M+H]⁺	714.548 4	0.0	C₄₀H₇₇NO₇P	715,656,184	PC (O-14∶0/18∶3)
45	9.20	[M+H]⁺	692.558 5	−1.3	C₃₈H₇₉NO₇P	693,184	PC (O-14∶0/16∶0)
46	9.24	[M−H]⁻	722.509 7	−3.9	C₄₁H₇₃NO₇P	722,301	PE (O-16∶0/20∶5)
47	9.24	[M−H]⁻	748.530 9	3.7	C₄₃H₇₅NO₇P	748,140	PE (O-16∶0/22∶6)
48	9.27	[M+H]⁺	746.570 9	1.2	C₄₁H₈₁NO₈P	746,184	PC (15∶0/18∶1)
		[M+CH₃COO]⁻	804.574 8	−0.9	C₄₃H₈₃NO₁₀P	804,730,241,281
49	9.40	[M+H]⁺	718.575 9	1.1	C₄₀H₈₁NO₇P	718,184	PC (O-32∶1)
50	9.54	[M+H]⁺	744.593 7	4.0	C₄₂H₈₃NO₇P	744,561,184	PC (O-16∶0/18∶2)
51	9.54	[M+H]⁺	772.588 6	3.9	C₄₃H₈₃NO₈P	772,184	PC (35∶2)
		[M+CH₃COO]⁻	830.595 0	4.7	C₄₅H₈₅NO₁₀P	830,279
52	9.59	[M+H]⁺	784.588 2	1.9	C₄₄H₈₃NO₈P	785,725,601,184	PC (16∶0/20∶3)
53	9.70	[M−H]⁻	778.576 8	2.2	C₄₅H₈₁NO₇P	778,140	PE (O-18∶0/22∶6)
54	9.70	[M+H]⁺	734.572 9	3.9	C₄₀H₈₁NO₈P	734,184	PC (16∶0/16∶0)
		[M+CH₃COO]⁻	792.574 8	−0.9	C₄₂H₈₃NO₁₀P	792,718,255
55	9.70	[M+H]⁺	760.588 6	3.9	C₄₂H₈₃NO₈P	760,522,496,478,184	PC (16∶0/18∶1)
		[M+CH₃COO]⁻	818.590 5	−0.7	C₄₄H₈₅NO₁₀P	818,744,462,281,255
56	9.70	[M+H]⁺	786.603 9	3.3	C₄₄H₈₅NO₈P	786,283,184	PC (18∶1/18∶1)
		[M+CH₃COO]⁻	844.610 0	3.8	C₄₆H₈₇NO₁₀P	844,281,168
57	9.87	[M+H]⁺	810.604 1	3.5	C₄₆H₈₅NO₈P	810,184	PC (18∶1/20∶3)
58	10.38	[M−H]⁻	716.525 0	2.8	C₃₉H₇₅NO₈P	716,283,253,196,140	PE (16∶0/18∶1)
59	10.38	[M−H]⁻	742.539 6	1.2	C₄₁H₇₇NO₈P	742,460,281,196,140	PE (18∶1/18∶1)
		[M+CH₃COO]⁻	802.556 6	−4.0	C₄₃H₈₁NO₁₀P	802,140
60	10.91	[M+H]⁺	768.555 3	−2.9	C₄₃H₇₉NO₈P	769,710,586,86	PC (15∶0/20∶4)
61	11.37	[M+H]⁺	720.590 5	1.9	C₄₀H₈₃NO₇P	721,184	PC (O-16∶0/16∶0)
62	11.77	[M+H]⁺	746.608 3	2.5	C₄₂H₈₅NO₇P	746,687,184	PC (O-16∶0/18∶1)
63	11.77	[M+H]⁺	772.623 5	1.9	C₄₄H₈₇NO₇P	772,713,184	PC (O-16∶0/20∶2)
64	11.77	[M+H]⁺	768.590 0	−0.9	C₄₄H₈₃NO₇P	768,709,184,86	PC (O-16∶0/20∶4)
65	11.77	[M+H]⁺	702.543 6	−0.3	C₃₉H₇₇NO₇P	702,561,283,141	PE (P-16∶0/18∶1)
		[M−H]⁻	700.526 1	−2.9	C₃₉H₇₅NO₇P	700,421,281,196,140
66	12.56	[M+H]⁺	810.599 8	2.2	C₄₆H₈₅NO₈P	811,752,628,184	PC (18∶1/20∶3)
67	12.89	[M+H]⁺	836.617 4	2.6	C₄₈H₈₇NO₈P	837,778	PC (18∶0/22∶5)
68	12.89	[M+H]⁺	762.600 5	−1.0	C₄₂H₈₅NO₈P	762,184	PC (34∶0)
69	13.10	[M+H]⁺	788.617 7	1.0	C₄₄H₈₇NO₈P	788,184	PC (18∶0/18∶1)
		[M+CH₃COO]⁻	846.621 9	−0.6	C₄₆H₈₉NO₁₀P	846,283,281
70	13.53	[M+H]⁺	814.633 9	1.7	C₄₆H₈₉NO₈P	814,184	PC (18∶0/20∶2)
71	13.70	[M+H]⁺	746.573 4	4.6	C₄₁H₈₁NO₈P	746,605	PE (18∶0/18∶1)
		[M−H]⁻	744.556 9	3.5	C₄₁H₇₉NO₈P	744,462,283,281,196,140
72	14.63	[M+H]⁺	748.623 9	2.5	C₄₂H₈₇NO₇P	748,689,184	PC (O-16∶0/18∶0)
73	15.10	[M+H]⁺	774.640 0	3.0	C₄₄H₈₉NO₇P	774,184	PC (P-20∶0/16∶0)
74	15.62	[M+H]⁺	756.594 5	5.0	C₄₃H₈₃NO₇P	756,184	PC (P-38∶2)
75	15.90	[M+H]⁺	790.635 4	3.5	C₄₄H₈₉NO₈P	790,184	PC (36∶0)
76	17.59	[M+H]⁺	802.671 6	3.2	C₄₆H₉₃NO₇P	802,184	PC (O-18∶0/20∶1)
77	17.59	[M+H]⁺	776.766 9	4.6	C₄₄H₉₁NO₇P	776,717,184	PC (O-36∶0)
78	18.49	[M+H]⁺	892.681 6	2.4	C₅₂H₉₅NO₈P	892,833,184	PC (44∶5)
79	18.55	[M+H]⁺	896.714 9	4.6	C₅₂H₉₉NO₈P	896,184	PC (44∶3)
80	18.58	[M+H]⁺	870.696 1	1.0	C₅₀H₉₇NO₈P	870,184	PC (42∶2)
81	18.77	[M+H]⁺	844.682 1	3.1	C₄₈H₉₅NO₈P	844,837,184	PC (16∶0/24∶1)
82	19.82	[M+H]⁺	812.654 6	1.6	C₄₇H₉₁NO₇P	812,671,337,141	PE (P-20∶0/22∶2)

Table 1 Identification of glycerophospholipids in RAW264.7 macrophage

2 甘油磷脂成分的质谱鉴定

在鉴定出的82个甘油磷脂成分中,包括PC类、PE类、PG类和PI类。PC类在正离子模式下,容易生成一个特征性的碎片离子,即m/z 184^[22]。在负离子模式下,由于流动相里含有乙酸,PC类容易形成 [M+ CH₃COO]⁻,也容易失去一个甲基形成 [M−CH₃]⁻^[23],此外,PC (16∶0/0∶0) 的脂肪酸链断裂形成碎片峰m/z 255,代表C16∶0脂肪酸链。

PE类在正离子模式下会形成准分子离子峰 [M+H]⁺,还可断裂极性的头部形成PE类在ESI⁺下二级串联质谱裂解的特征峰 [M+H−141]⁺^[24]。在负离子模式下,PE类会形成准分子离子峰 [M−H]⁻,还可裂解产生PE类物质在负离子模式下的特征峰m/z 140 ([C₂H₇O₄NP]⁻) 和m/z 196([C₅H₁₁O₅NP]⁻)^[25],此外,PE (18∶0/0∶0) 的脂肪酸链断裂形成碎片峰m/z 283,代表C18∶0脂肪酸链。

PI类在负离子模式下,会形成准分子离子峰 [M−H]⁻,此外,其极性头部的不同断裂还会产生一些特征峰,如果断裂sn-1位置的链,将产生m/z 223 ([C₆H₈O₇P]⁻) 和m/z 297 ([C₉H₁₄O₉P]⁻)^[26],如果sn-2位置的脂肪酸链断裂形成乙烯酮结构,将产生m/z 241 ([C₆H₁₀O₈P]⁻)、m/z 223 ([C₆H₈O₇P]⁻) 和m/z 315 ([C₉H₁₆O₁₀P]⁻),这些特征性的离子碎片即可判别生物样品中是否存在PI类物质^{[26, 27]}。另外,PI (16∶0/ 18∶1) 的两条脂肪酸链分别断裂形成m/z 253和m/z 281,代表C16∶1和C18∶1两条脂肪酸链,且m/z 281的信号强度比m/z 253强,说明C18∶1键合在甘油骨架的sn-2位^[28]。

PG类在负离子模式下断裂一条脂肪酸链形成[M−H−R₂COOH]⁻,在此基础上,还会继续断裂形成[M−H−R₂COOH−74]⁻^[29],另外,PG类容易裂解产生m/z 171 ([C₃H₈O₆P]⁻) 和m/z 227 ([C₆H₁₂O₇P]⁻) 的特征峰^[25]。

本实验通过运用UHPLC-QTOF/MS技术并结合各类甘油磷脂类裂解规律,共推断出82个甘油磷脂成分,其中包括57个磷脂酰胆碱 (PCs)、21磷脂酰乙醇胺 (PEs)、3个磷脂酰甘油 (PGs) 和1个磷脂酰肌醇 (PI)。本实验表明UHPLC-QTOF/MS能够有效地分离推断RAW264.7巨噬细胞甘油磷脂成分。因此,本文建立了一种基于UHPLC-QTOF/MS技术的快速、简便、可靠的RAW264.7巨噬细胞甘油磷脂类成分快速表征方法,为以RAW264.7巨噬细胞作为研究对象的脂质组学研究提供依据。

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药学学报 2016, Vol. 51 Issue (9): 1451-1457	PDF