UPLC-ESI-MS分析中棉花次生代谢物标准品推定和加合物形成

李社增,牛露欣,李博超,陈秀叶,马平,马峙英

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棉花学报 ›› 2020, Vol. 32 ›› Issue (5) : 449-462. DOI: 10.11963/1002-7807.lszmzy.20200723
研究简报

UPLC-ESI-MS分析中棉花次生代谢物标准品推定和加合物形成

作者信息 +

Putative Identification and Adduct Formation of Reference Materials for Cotton Secondary Metabolite using UPLC-ESI-MS Analysis

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摘要

【目的】 建立超高效液相色谱电喷雾质谱（Ultra-performance liquid chromatography-electrospray ionization-mass spectrometry,UPLC-ESI-MS）分析中棉花代谢产物的高通量鉴定方法;探讨特定UPLC和ESI模式条件下棉花次生代谢产物的加合物种类、主导加合物及适宜的ESI模式。【方法】 利用高效液相色谱电喷雾质谱对18个棉花代谢产物标准品进行分析,采用在线XCMS软件进行无靶标质谱数据提取,利用MATLAB软件编程计算程序建立标准品鉴定方法。【结果】 针对电喷雾正、负离子模式,建立了基于计算准确相对分子质量的棉花次生代谢产物标准品的快速鉴定方法POSid和NEGid;特定超高效液相色谱和电喷雾正、负离子模式下14个标准品得到正确鉴定。正离子模式下出现[M+H]+、[M+Na]+、[M+NH4]+、[2M+NH4]+、[2M+Na]+和[2M+H]+ 6种加合物,负离子模式下出现[M-H]-、[2M-H]-、[M+Cl]-、[M+FA-H]-、[3M-H]-、[M+Na-2H]-、[M-H2O-H]-和[M+TFA-H]- 8种加合物,单个标准品的质谱可观察到1~6种加合物,每个标准品均有主导加合物并具有电喷雾离子模式的偏好性。蜜二糖适合电喷雾正离子模式检测,棉酚适合2种离子模式检测;12种化合物均是负离子模式信号强于正离子模式,适合采用负离子模式检测。【结论】 建立的基于计算准确相对分子质量的代谢产物鉴定方法,能实现对18个棉花次生代谢产物标准品的无靶标质谱数据的鉴定。特定超高效液相色谱和电喷雾条件下,棉花次生代谢产物的主导加合物具有电喷雾离子模式的偏好性。这些结果为开展棉花代谢组研究提供了技术和理论数据支撑。

Abstract

[Objective] The aim of this study is to establish an method for cotton metabolites identification by ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) analysis, and to investigate the adduct types, dominant adducts and appropriate ESI ion modes of cotton secondary metabolites under the determined UPLC and ESI conditions. [Method] UPLC-ESI-MS was employed to analyze 18 cotton metabolite standards, Online XCMS software was used to extract the nontargeted mass spectrum data, and MATLAB software was used to prepare calculation programs for the identification method of cotton metabolite standards. [Result] A high-throughput identification method of cotton secondary metabolite, named POSid and NEGid separately for the positive and negative ESI modes, was established based on the calculated accurate molecular weight. In the determined UPLC condition and positive and negative ESI mode, 14 cotton metabolite standards were correctly identified. There found 6 adducts including [M+H]+, [M+Na]+, [M+NH4]+, [2M+NH4]+, [2M+Na]+ and [2M+H]+ in positive ion mode, and 8 adducts including [M-H]-, [2M-H]-, [M+Cl]-, [M+FA-H]-, [3M-H]-, [M+Na-2H]-, [M-H2O-H]- and [M+TFA-H]- in negative ion mode, while 1 to 6 adducts were observed in the mass spectrum of a single standard, and each metabolite standard had a dominant adduct of a preference for ESI mode. Melibose was suitable for ESI positive ion mode detection, gossypol was suitable for both ion modes detection; and 12 compounds were suitable for negative ion mode detection due to mass spectrum signals of their dominant adduct stronger in negative ion mode than positive ion mode. [Conclusion] Based on the accurate molecular weight, the established identifying method can identify the 18 cotton metabolite standards with their nontarget mass spectrometry data. Under the determined UPLC and ESI condition, the dominant adducts of cotton secondary metabolites have the preference of ESI mode. These results provide technical and theoretical data support for further study of cotton metabolomics.

关键词

棉花次生代谢产物 / 超高效液相色谱-电喷雾质谱 / 质谱鉴定 / 加合物

Keywords

cotton metabolite / ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) / mass spectrum identification / adduct

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李社增 , 牛露欣 , 李博超 , 陈秀叶 , 马平 , 马峙英. UPLC-ESI-MS分析中棉花次生代谢物标准品推定和加合物形成[J]. 棉花学报, 2020, 32(5): 449-462. https://doi.org/10.11963/1002-7807.lszmzy.20200723
Li Shezeng , Niu Luxin , Li Bochao , Chen Xiuye , Ma Ping , Ma Zhiying. Putative Identification and Adduct Formation of Reference Materials for Cotton Secondary Metabolite using UPLC-ESI-MS Analysis[J]. Cotton Science, 2020, 32(5): 449-462. https://doi.org/10.11963/1002-7807.lszmzy.20200723
采用气相色谱-质谱（Gas chromatography-mass spectrometer,GC-MS）[1,2]或液相色谱-质谱（Liquid chromatography-mass spectrometer,LC-MS）[3,4]成功分析拟南芥、番茄等植物代谢组后,色谱与质谱相结合分析技术被证明是非靶标代谢组分析中最高效和最灵敏的方法。在非靶标代谢组研究中,由于质谱数据庞大而复杂,数据处理和分析以及随后的代谢物注释/鉴定是最耗时费力的工作。造成代谢组数据庞大复杂的主要原因是分析物的多种加合物形成以及盐团簇形成,导致质谱数据存在大量的额外信号[5]。
在液相色谱电喷雾质谱（Liquid chromatography-electrospray ionization-mass spectrometry,LC-ESI-MS）分析过程中,分析物形成加合物的现象普遍存在,是对物质定性、定量的基础[6,7,8]。尤其对于糖和各种炸药等一些不能直接用电喷雾质谱分析的化合物,加合物形成的发现显得尤为重要。例如：Ghosh等[9]在电喷雾负离子模式下通过甲酸加合物实现了酰基蔗糖代谢物的测定,Mathis等[10]采用负离子加合物建立了高能炸药的定性和定量的方法。加合物中除了常见的质子化分子外,钠、钾和铵加合物也经常出现,Huang等[11]报道在电喷雾质谱分析中的正（负）离子模式中能够观察到常见的正、负离子加合物分别为32个和15个。而与分析物一起形成加合物的各种离子,一般来源于液相色谱的流动相及其添加剂、溶剂杂质、玻璃器皿等[12,13,14],特别是流动相中加入添加剂对加合物形成具有较大的影响。Kruve等[15]研究了ESI正离子模式下7种流动相添加剂对17个化学物质加合物形成的可能性,明确流动相添加剂改变加合物形成有很强的作用;Erngren等[16]的研究证明流动相中钠离子或钾离子浓度对加合物的定量影响极大,甚至形成多聚加合物;Leitner等[17]研究6种不同的乙腈/醋酸铵混合物在中性条件下对模型肽（缓激肽）高效液相色谱（High performance liquid chromatography,HPLC）-ESI-MS分析的性能,明确流动相改变对色谱分离和加合物的形成影响很大,在供试的条件下,可以形成钠化肽加合物而不是常见的质子加合物。因此,对于特定的化合物,明确特定液相色谱和电喷雾离子条件下加合物形成数据,对该物质的定量和定性分析是非常必要的。
在采用LC-ESI-MS的代谢组分析中,明确代谢产物的种类尤为重要。由于LC-ESI-MS系统的多样性以及液相色谱较低的保留时间再现性,导致单一优化分析方法和液相色谱-质谱分析中的色谱图和质谱图在不同实验室间进行比较均受到严重限制,同时还缺少将质谱数据自动转换为（推定的）植物代谢物的高效工具。这些问题导致对大量质谱信号数据的分析只能在现有的化学数据库（如SciFinder、Pub-Chem、Massbank或Dictionary of Natural Products）中进行手动搜索[18],筛选效率受到极大限制,同时这些数据库的信息来自一般的化学物质,其来源未与植物关联,导致植物代谢组鉴定时推定的物质种类存在多种可能性和不准确的靶标。因此,如何实现代谢产物准确鉴定是代谢组分析中的主要难题。在线XCMS软件在一定程度上解决了不同型号液相色谱-质谱系统采集的数据处理,实现特征离子检测、保留时间校正、峰对齐注释、统计分析和数据可视化的自动化,为完整的非靶标代谢组分析提供了一整套解决方案[19,20]。然而,作者所在实验室在前期研究中,发现通过在线XCMS软件进行代谢产物鉴定,推定的化合物与报道的棉花代谢产物存在较大差异,所以该软件在针对具体植物的代谢产物进行鉴定时存在重大不足。
本研究着重针对在线质谱分析技术不能准确鉴定棉花次生代谢产物的科学问题,根据LC-ESI-MS分析中正（负）离子模式下形成加合物的一般规律,利用MATLAB软件编写计算程序建立棉花代谢产物快速鉴定方法;同时,利用超高效液相色谱电喷雾质谱（Ultra-performance liquid chromatography-electrospray ionization-mass spectrometry,UPLC-ESI-MS）方法对棉花代谢产物标准品进行质谱分析,采用在线XCMS软件进行无靶标质谱数据提取并利用本研究建立的鉴定方法进行鉴定,明确特定液相色谱和电喷雾离子模式条件下这些棉花代谢产物的加合物种类、主导加合物及适宜的离子检测模式。因此,本研究结果将为棉花次生代谢组学研究提供技术和理论数据支撑。

1 材料与方法

1.1 棉花次生代谢产物标准品检测液制备

对18个棉花代谢产物的标准品（表1）开展UPLC-ESI-MS分析研究。称取1.0 mg单个标准品加入10.0 mL适用溶剂进行溶解,制备标准品100.0 mg·L-1母液。取单个标准品母液0.1 mL,用甲醇作稀剂释定容至10 mL,混匀后制备成质量浓度为1.0 mg·L-1的溶液,作为单个标准品检测液;分别取18个标准品的母液各0.1 mL于容量瓶中进行混合,用甲醇作稀释剂定容至10 mL,混匀后制备成单个标准品质量浓度为1.0 mg·L-1的标准品混合检测液,用于液相色谱-质谱分析的质量控制检测。这2种检测液分别分装于进样瓶中并储存在4 ℃冰箱中备用。
表1 棉花代谢产物标准品的信息

Table 1 Information on cotton metabolite standards tested in presented study

化合物
Compound		 单同位素准确相对分子质量
Monoisotopic molecular weight		 分子式
Molecular Formula		 CAS号
CAS number		 分类
Type		 适用溶剂
Suitable solvent		 生产商
Manufacturer
赤霉素A4
Gibberellin A4		 332.162 374 C19H24O5 468-44-0 二萜类
Diterpenoids		 甲醇
Methanol		 TLC Pharmaceutical Standards
赤霉素A3
Gibberellin A3		 346.141 638 C19H22O6 77-06-5 二萜类
Diterpenoids		 丙酮
Acetone		 北京北方伟业计量技术研究院
Beijing North Weiye Measurement Technology Research Institute
水杨酸
Salicylic acid		 138.031 694 C7H6O3 69-72-7 酚类
Phenols		 二甲基亚砜
Dimethylsulfoxide (DMSO)		 上海源叶生物科技有限公司
Shanghai Yuanye Bio-
Technology Co., Ltd (SYBT Co. Ltd)
柽柳黄素
Tamarixetin		 316.058 305 C16H12O7 603-61-2 类黄酮
Flavonoids		 二甲基亚砜
DMSO		 SYBT Co. Ltd
N6-异戊烯基腺嘌呤N6-(delta2-isopentenyl) adenine 203.117 096 C10H13N5 2365-40-4 生物碱类
Alkaloids		 二甲基亚砜
DMSO		 SYBT Co. Ltd
化合物
Compound		 单同位素准确相对分子质量
Monoisotopic molecular weight		 分子式
Molecular Formula		 CAS号
CAS number		 分类
Type		 适用溶剂
Suitable solvent		 生产商
Manufacturer
α-石竹烯
α-humulene		 204.187 801 C15H24 6753-98-6 石竹烷倍半萜类Caryophyllane sesquiterpenoids 乙醇
Ethanol		 SYBT Co. Ltd
角鲨烯
Squalene		 410.391 252 C30H50 7683-64-9 线性三萜类
Linear triterpenoids;		 乙醇
Ethanol		 SYBT Co. Ltd
蜜二糖
Melibiose		 342.116 215 C12H22O11 585-99-9 糖类
Carbohydrates		 去离子水
Deionized water		 SYBT Co. Ltd
蔗糖
Sucrose		 342.116 215 C12H22O11 585-99-9 糖类
Carbohydrates		 去离子水
Deionized water		 SYBT Co. Ltd
紫云英苷
Astragalin		 448.100 562 C21H20O11 480-10-4 类黄酮类
Flavonoids		 去离子水
Deionized water		 SYBT Co. Ltd
脱落酸
(s)-(+)-abscisic acid		 264.136 159 C15H20O4 21293-29-8 环法尼烷倍半
Cyclofarnesane sesquiterpenoids		 甲醇
Methanol		 SYBT Co. Ltd
绿原酸3-O-caffeoylquinic acid 354.095 082 C16H18O9 327-97-9 酚类
Phenols		 甲醇
Methanol		 SYBT Co. Ltd
白麻苷Quercetin 3-sophoroside 626.148 305 C27H30O17 18609-17-1 类黄酮类
Flavonoids		 甲醇
Methanol		 SYBT Co. Ltd
咖啡酸
Caffeic acid		 180.042 259 C9H8O4 501-16-6 酚类
Phenols		 甲醇
Methanol		 SYBT Co. Ltd
青榆烯C
Lacinilene C		 246.125 595 C15H18O3 41653-72-9 杜松烷倍半萜
Cadinane sesquiterpenoids		 甲醇
Methanol		 SYBT Co. Ltd
3-吲哚甲醛1H-indole-3-carboxaldehyde 145.052 764 C9H7NO 487-89-8 生物碱类
Alkaloids		 甲醇
Methanol		 SYBT Co. Ltd
棉酚
Gossypol		 518.194 068 C30H30O8 303-45-7 杜松烷倍半萜 Cadinane sesquiterpenoids 甲醇
Methanol		 SYBT Co. Ltd
三十烷酸
Triacontanoic acid		 452.459 331 C30H60O2 506-50-3 脂肪族天然产物 Aliphatic natural products 甲醇
Methanol		 SYBT Co. Ltd

1.2 液相色谱-质谱检测

本研究采用超高效液相色谱电喷雾飞行时间质谱仪（UPLC型号为Nexera LC-30AD,日本岛津制作所制造;质谱仪为Triple TOFTM 5600+,美国AB SCIEX公司生产）对棉花代谢产物标准品及其混合检测液进行分析。
色谱条件：色谱柱为Shim-pack GIST C18（直径2.1 mm,长100 mm,填料粒径2 μm）,流动相A相为0.1%（体积分数）甲酸水溶液,流动相B相为0.1%（体积分数）甲酸乙腈溶液,流速0.35 mL·min-1,柱温40 ℃,自动进样器温度4 ℃,进样量4 μL。每个样品3次重复。洗脱条件：0~1.0 min,5%（体积分数,下同）B;1.0~6.0 min,5%~20%B;6.0~9.0 min,20%~50%B;9.0~13.0 min,50%~95%B;13.0~15.0 min,95% B;15.0~15.2 min,95%~5%B;15.2~17.5 min,5%B。
正离子模式（Positive ion mode,ESI+）质谱检测：喷雾气压（GAS1）344.7 kPa（50 psi）,辅助加热气压（GAS2）413.7 kPa（60 psi）,辅助加热气温度500 ℃,气帘气压（CUR）241.3 kPa（35 psi）,离子化电压（IS）5 500 V,采集模式为信息依赖型采集（IDA）模式,TOF-MS 质荷比（m/z）扫描范围50~1 500,累积时间250 ms,TOF-MS/MS二级m/z扫描范围50~1 000,累积时间50 ms,去簇电压（DP）100 V,碰撞能量（CE）35 eV,扩展碰撞能量（CES）15 eV。
负离子模式（Negative ion mode,ESI-）质谱检测：辅助加热气压379.2 kPa（55 psi）,气帘气压172.4 kPa（25 psi）,离子化电压4 500 V,其他参数设置与正离子模式质谱检测一致。
采用甲酸钠相对分子质量作为锁定值,对质谱系统的稳定性及准确性进行校正,每6个样品自动校正1次。

1.3 质谱数据预处理和分析

采用在线XCMS软件对UPLC-ESI-TOF/MS数据进行可视化和手工处理（ https://xcmsonline.scripps.edu）[20],进行特征离子峰提取、保留时间（Retention time,RT）校正和峰对齐,获得m/z、保留时间和峰强度（Peak intensity）等数据,进一步将包含RT、m/z和峰强度等数据导出为xlsx文件,在MS Excel 2010软件中手动搜索和编辑,包括杂质峰的消除和数据去噪。将最终结果转化为数据矩阵（RT×m/z×峰强度）,分别构建ESI+变量数据集和ESI-变量数据集,并用于进一步的数据分析。

1.4 棉花谢产物的鉴定计算程序

根据LC-ESI-MS分析技术中正（负）离子模式下形成加合物的一般规律,利用MATLAB软件（R2018b, The MathWorks, Inc.）编写计算程序,根据每个离子的质荷比计算出对应的所有可能的准确相对分子质量（计算相对分子质量）,进一步计算其与各标准品准确相对分子质量间的误差,根据锁定相对分子质量检测误差设定误差阈值,以介于此阈值正负值间的推定物质作为供试化合物的鉴定结果。

1.5 UPLC-ESI-MS检测中棉花代谢产物鉴定及加合物推定

根据方法1.4中对棉花次生代谢产物标准品的质谱数据鉴定结果,调查供试标准品对应的质谱离子形态,即为UPLC-ESI-MS检测中棉花代谢产物标准品的加合物。

1.6 UPLC-ESI-MS检测中离子模式对棉花代谢产物信号影响

筛选正离子模式和负离子模式下各标准品的信号最强的主导加合物,计算2个离子模式下峰强度的比值。

2 结果与分析

2.1 标准品的UPLC-ESI-TOF/MS检测数据

18个标准品混合检测液的UPLC-ESI-MS总离子色谱图见图1。图1显示代谢产物脱落酸,在2个模式中保留时间和峰强度均有所不同,在正离子模式下,其主要加合物[M+H]+（m/z 265.142 9）保留时间为9.96 min,峰强度为1.76×105（图1A）,负离子模式下主要加合物[M-H]-（m/z 263.129 4）保留时间为10.01 min,峰强度为5.62×105（图1B）。采用在线XCMS软件,分别对正离子模式和负离子模式下采集的各个标准品的原始质谱数据进行处理和无靶标离子提取,数据导出到xlsx文件中,为每个标准品均构建了ESI+数据集和ESI-数据集。导出的数据中包含了各离子的保留时间、m/z和峰强度。每个标准品提取到的离子数量、m/z范围、保留时间不同,详细情况列于表2。
图1 正离子模式（A）和负离子模式（B）下棉花代谢物标准品混合液UPLC-ESI-MS 总离子色谱图

Fig. 1 UPLC-ESI-MS TICs of cotton metabolite mixture in positive (A) and negative (B) ion mode

Full size|PPT slide

表2 棉花代谢产物标准品的UPLC-ESI-MS检测数据概况

Table 2 A survey of UPLC-ESI-MS data of cotton metabolite standards

化合物
Compound		 负离子模式 Positive ion mode 负离子模式Negative ion mode
离子数
Amount of ions		 质荷比
m/z		 保留时间
Retention time/min		 离子数
Amount of ions		 质荷比
m/z		 保留时间
Retention time/min
水杨酸 Salicylic acid 2 165 59.052 0~1 431.294 3 0.13~17.47 1 523 61.993 2~1 405.012 8 0.68~17.44
柽柳黄素 Tamarixetin 2 165 59.052 0~1 431.294 3 0.13~17.47 1 523 61.993 2~1 405.012 8 0.68~17.44
N6-异戊烯基腺嘌呤
N6-(delta2-isopentenyl) adenine		 1 977 59.052 1~1 431.796 0 0.22~17.47 1 560 61.993 4~1 418.991 2 0.68~17.45
α-石竹烯 α-humulen 1 410 59.055 6~1 222.341 8 0.14~17.47 1 225 61.991 6~1 405.003 6 0.69~17.44
角鲨烯Squalene 1 480 60.083 7~1 223.338 3 0.70~17.47 1 149 61.993 1~1 470.000 9 0.16~17.47
蜜二糖 Melibiose 1 945 59.055 4~1223.3365 0.14~17.47 1 797 61.990 3~1 487.099 2 0.16~17.48
蔗糖 Sucrose 1 945 59.055 4~1 223.336 5 0.14~17.47 1 797 61.990 3~1 487.099 2 0.16~17.48
紫云英苷 Astragalin 2 071 59.055 4~1 431.894 0 0.14~17.47 1 709 61.990 1~1 487.099 2 0.16~17.48
脱落酸 (s)-(+)-abscisic acid 1 920 59.055 4~1 431.791 4 0.14~17.47 1 450 61.990 1~1 488.096 8 0.16~17.48
绿原酸
3-O-caffeoylquinic acid		 1 920 59.055 4~1 431.791 4 0.14~17.47 1 450 61.990 1~1 488.096 8 0.16~17.48
白麻苷
Quercetin 3-sophoroside		 1 668 60.083 2~1 431.792 1 0.23~17.47 1 406 61.992 7~1 488.096 8 0.18~14.48
咖啡酸 Caffeic acid 1 997 59.055 7~1 431.791 4 0.14~17.47 1 451 61.990 1~1 488.096 8 0.16~17.48
青榆烯C Lacinilene C 1 957 59.055 5~1 431.791 4 0.14~17.47 1 522 61.993 8~1 488.096 8 0.70~17.47
3-吲哚甲醛1H-
indole-3-carboxaldehyde		 1 670 60.083 2~1 431.791 4 0.74~17.47 1 421 61.992 8~1 488.096 8 0.68~17.48
赤霉素A4 Gibberellin A4 1 694 60.083 3~1 431.791 4 0.74~17.47 1 612 61.990 4~1 488.096 8 0.18~14.48
棉酚 Gossypol 1 936 60.083 4~1 431.791 4 0.74~17.47 1 481 61.993 7~1 488.096 8 0.68~14.48
赤霉素A3 Gibberellin A3 1 911 60.082 7~1 431.791 4 0.05~17.47 1 574 61.990 5~1 488.096 8 0.18~14.47
三十烷酸
Triacontanoic acid		 1 647 59.055 3~1 150.319 2 0.14~17.48 911 61.993 1~1 403.001 7 0.61~17.45

2.2 化合物鉴定的推定方法

在LC-ESI-MS分析中分析物最终形成常见的正、负离子模式加合物分别为32个和15个,根据这些加合物构成规律,设计了根据加合物的质荷比计算准确相对分子质量的公式,列于表3。本研究利用MATLAB软件编制2个计算程序POSid和NEGid（暂不公开）,分别对正离子模式和负离子模式下检测到的质谱数据进行计算,给出判定结果。这个计算过程主要包括3个步骤：（1）获得计算的准确相对分子质量（Calculated molecular weight,CM）：根据各检测到离子的m/z,计算出每个离子对应的所有可能的准确相对分子质量,计算公式见表3;（2）获得误差（ΔM）：计算各离子的计算准确分子量与各标准品准确相对分子质量（MM,表1）间的误差,计算公式：ΔM=（CM-MM）/MM;（3）根据相对分子质量锁定值的检测误差设定误差阈值,以误差介于此阈值正负值的推定物质作为供试物质的鉴定结果。
表3 LC-ESI-MS检测中供试物质的常见加合物

Table 3 Common adducts of the tested compound in LC-ESI-MS detection

正离子模式Positive ion mode 负离子模式 Negative ion mode
加合物名称a
Adduct name		 计算的准确相对分子质量b
Calculated molecular weight (CM)		 加合物名称a
Adduct name		 计算的准确相对分子质量b
Calculated molecular weight (CM)
[M+3H]3+ 3×（m/z-1.007 276） [M-3H]3- 3×（m/z+1.007 276）
[M+2H+Na]3+ 3×（m/z-8.334 590） [M-2H]2- 2×（m/z+1.007 276）
[M+H+2Na]3+ 3×（m/z-15.7 661 904） [M-H2O-H]- m/z+19.01 839
[M+3Na]3+ 3×（m/z-22.989 218） [M-H]- m/z+1.007 276
[M+2H]2+ 2×（m/z-1.007 276） [M+Na-2H]- m/z-20.974 666
[M+H+NH4]2+ 2×（m/z-9.520 550） [M+Cl]- m/z-34.969 402
[M+H+Na]2+ 2×（m/z-11.998 247） [M+K-2H]- m/z-36.948 606
[M+H+K]2+ 2×（m/z-19.985 217） [M+FA-H]- m/z-44.998 201
[M+ACN+2H]2+ 2×（m/z-21.520 550） [M+HAc-H]- m/z-59.013 851
[M+2Na]2+ 2×（m/z-22.989 218） [M+Br]- m/z-78.918 885
[M+2ACN+2H]2+ 2×（m/z-42.033 823） [M+TFA-H]- m/z-112.985 586
[M+3ACN+2H]2+ 2×（m/z-62.547 097） [2M-H]- （m/z+1.007 276）÷2
[M+H]+ m/z-1.007 276 [2M+FA-H]- （m/z-44.998 201）÷2
[M+NH4]+ m/z-18.033 823 [2M+HAc-H]- （m/z-59.013 851）÷2
[M+Na]+ m/z-22.989 218 [3M-H]- （m/z+1.007 276）÷3
[M+CH3OH+H]+ m/z-33.033 489
[M+K]+ m/z-38.963 158
[M+ACN+H]+ m/z-42.033 823
[M+2Na-H]+ m/z-44.971 160
[M+IsoProp+H]+ m/z-61.06 534
[M+ACN+Na]+ m/z-64.015 765
[M+2K-H]+ m/z-76.919 040
[M+DMSO+H]+ m/z-79.02 122
[M+2ACN+H]+ m/z-83.060 370
[M+IsoProp+Na+H]+ m/z-84.05 511
[2M+H]+ （m/z-1.007 276）÷2
[2M+NH4]+ （m/z-18.033 823）÷2
[2M+Na]+ （m/z-22.989 218）÷2
[2M+3H2O+2H]+ （m/z-28.02 312）÷2
[2M+K]+ （m/z-38.963 158）÷2
[2M+ACN+H]+ （m/z-42.033 823）÷2
[2M+ACN+Na]+ （m/z-64.015 765）÷2
注：aM,物质分子;ACN,乙腈;DMSO,二甲基亚砜;FA,甲酸;HAc,乙酸;TFA,三氟乙酸;IsoProp,异丙醇;CH3OH,甲醇;b 由准确相对分子质量推导相应质荷比的计算公式。
Note: aM, compound molecular; ACN, acetonitrile; DMSO, dimethylsulfoxide; FA, formic acid; HAc, acetic acid; TFA, trifluoroacetic acid; IsoProp, isopropanol; CH3OH, methanol. b The formula for calculated molecular weight derived from m/z in this column.

2.3 棉花代谢产物鉴定及加合物形成

利用2.2中鉴定方法,根据检测到各标准品离子的m/z,计算了UPLC-ESI-MS检测到的各离子对应的所有可能的准确相对分子质量及其与各标准品的准确相对分子质量间的误差;本次检测锁定相对分子质量试验误差为21×10-6,确定20×10-6为误差阈值进行了化合物推定。对正（负）离子模式下检测到的离子,该鉴定方法能分别推定出14个标准品,但均未推定到α-石竹烯、角鲨烯和三十烷酸3种物质,另外正离子模式未推定到3-吲哚甲醛,负离子模式未推定到青榆烯C。在推定的化合物中,质谱观察到1~6种不同的加合物,峰强度5 600以上,正离子模式的误差-2.9~3.1,负离子模式误差-13.5~16.8（表4和表5）。
表4 正离子模式下棉花代谢产物标准品鉴定

Table 4 Putative identification of cotton metabolite standards in positive ion mode

化合物Compound 质荷比
m/z		 保留时间
Retention time/min		 加合物
Adduct		 计算的相对分子质量
Calculated molecular weight (CM)		 误差
Error (ΔM）/
10-6		 峰强度（均值±标准差）
Peak intensity (mean±standard deviation)
水杨酸 Salicylic acid 139.039 372 9.40 [M+H]+ 138.032 096 -2.9 7 978.8±695.4
柽柳黄素 Tamarixetin 317.064 865 10.99 [M+H]+ 316.057 589 2.3 79 834.0±2 961.4
N6-异戊烯基腺嘌呤
N6-(delta2-isopentenyl) adenine		 204.124 352 7.52 [M+H]+ 203.117 076 0.1 122 130.8±3 291.5
蜜二糖Melibiose 365.104 807 0.82 [M+Na]+ 342.115 589 1.8 109 513.7±5 559.8
360.149 727 [M+NH4]+ 342.115 904 0.9 209 629.9±6 917.8
343.123 083 [M+H]+ 342.115 807 1.2 12 543.4±919.6
蔗糖 Sucrose 365.104 807 0.82 [M+Na]+ 342.115 589 1.8 29 171.7±676.7
360.149 727 [M+NH4]+ 342.115 904 0.9 63 866.2±4 974.6
343.123 083 [M+H]+ 342.115 807 1.2 11 979.9±519.5
紫云英苷 Astragalin 471.088 494 9.14 [M+Na]+ 448.099 276 2.9 11 338.0±362.1
449.106 679 [M+H]+ 448.099 403 2.6 70 894.7±2 835.8
脱落酸 (s)-(+)-abscisic acid 287.124 907 10.05 [M+Na]+ 264.135 689 1.8 10 936.9±945.2
265.143 275 [M+H]+ 264.135 999 0.6 26 010.4±499.2
546.305 596 10.03 [2M+NH4]+ 264.135 887 1.0 14 020.0±420.6
551.260 231 [2M+Na]+ 264.135 506 2.5 25 030.7±1 001.2
529.278 663 [2M+H]+ 264.135 694 1.8 17 756.3±754.8
282.169 968 [M+NH4]+ 264.136 145 0.1 6 672.3±333.6
绿原酸 3-O-caffeoylquinic acid 355.102 131 6.22 [M+H]+ 354.094 855 0.6 85 454.1±4272.7
377.083 865 [M+Na]+ 354.094 537 1.2 19 178.5±392.8
377.083 755 6.94 [M+Na]+ 354.094 725 1.5 12 596.9±894.4
355.102 001 [M+H]+ 354.094 647 1.0 35 417.9±137.6
372.128 286 [M+NH4]+ 354.094 463 1.7 20 344.4±1 831.0
白麻苷 Quercetin 3-sophoroside 649.136 524 7.98 [M+Na]+ 626.147 306 1.6 7 629.1±177.2
627.154 853 [M+H]+ 626.147 577 1.2 113 288.4±6 797.3
咖啡酸Caffeic acid 181.049 082 6.79 [M+H]+ 180.041 806 2.5 14 140.3±289.6
青榆烯C Lacinilene C 247.132 676 12.50 [M+H]+ 246.125 400 0.8 514 004.6±20 560.2
510.284 037 [2M+NH4]+ 246.125 107 2.0 22 745.4±88.4
515.239 456 [2M+Na]+ 246.125 119 1.9 146 961.4±4 408.8
赤霉素A4 Gibberellin A4 333.169 188 11.19 [M+H]+ 332.161 912 1.4 7 749.0±180.0
棉酚 Gossypol 519.199 742 14.63 [M+H]+ 518.192 466 3.1 334 371.6±20 062.3
赤霉素A3 Gibberellin A3 347.148 415 8.56 [M+H]+ 346.141 139 1.4 6 354.0±24.7
364.175 788 8.57 [M+NH4]+ 346.141 638 -0.9 10 071.32±906.4
表5 负离子模式下棉花代谢产物标准品鉴定

Table 5 Putative identification of cotton metabolite standards in negative ion mode

化合物Compound 质荷比
m/z		 保留时间
Retention time/min		 加合物
Adduct		 计算的相对分子质量
Calculated molecular weight (CM)		 误差
Error (ΔM）/
10-6		 峰强度（均值±标准差）
Peak intensity (mean±standard deviation)
水杨酸Salicylic acid 137.025 536 9.43 [M-H]- 138.032 812 -8.1 198 088.9±5 942.7
柽柳黄素Tamarixetin 631.108 934 10.95 [2M-H]- 316.058 105 0.6 109 455.2±4 060.1
315.051 258 10.95 [M-H]- 316.058 534 -0.7 816 319.8±22 000.2
N6-异戊烯基腺嘌呤
N6-(delta2-isopentenyl) adenine		 202.110 473 7.54 [M-H]- 203.117 749 -3.2 189 001.7±9 595.2
341.109 546 0.84 [M-H]- 342.116 822 -1.8 24 146.5±796.8
蜜二糖Melibiose
387.114 900 0.84 [M+FA-H]- 342.116 699 -1.4 32 762.2±2 401.8
蔗糖Sucrose 341.109 546 0.84 [M-H]- 342.116 822 -1.8 279 325.5±6 480.0
387.114 900 0.84 [M+FA-H]- 342.116 699 -1.4 315 114.6±24 544.5
紫云英苷Astragalin 447.092 879 9.19 [M-H]- 448.100 155 0.9 706 162.4±30 621.1
895.194 237 9.19 [2M-H]- 448.100 757 -0.4 61 671.9±1 969.9
脱落酸
(s)-(+)-abscisic acid		 527.262 734 10.04 [2M-H]- 264.135 005 4.4 820 955.1±32 838.2
263.129 076 10.04 [M-H]- 264.136 352 -0.7 566 054.7±5 660.5
299.104 968 10.06 [M+Cl]- 264.135 566 2.2 11 299.5±216.9
绿原酸3-O-caffeoylquinic acid 353.086 885 6.10 [M-H]- 354.094 161 2.6 160 270.3±4 808.1
707.181 308 6.18 [2M-H]- 354.094 292 2.2 8 307.3±332.3
375.067 942 6.18 [M+Na-2H]- 354.093 276 5.1 6 738.5±286.4
353.087 136 6.90 [M-H]- 354.094 412 1.9 214 170.1±4 283.4
707.181 680 6.90 [2M-H]- 354.094 478 1.7 12 790.6±639.5
375.068 452 6.90 [M+Na-2H]- 354.093 786 3.7 17 940.0±367.4
白麻苷
Quercetin 3-sophoroside		 625.140 555 7.98 [M-H]- 626.147 831 0.8 1 433 724.8±37 276.8
661.117 307 7.98 [M+Cl]- 626.147 905 0.6 171 675.8±667.0
1251.287805 7.98 [2M-H]- 626.147 541 1.2 20 615.0±1 855.4
739.123 388 7.99 [M+TFA-H]- 626.137 802 16.8 7 925.1±184.1
咖啡酸Caffeic acid 179.035 573 6.71 [M-H]- 180.042 849 -3.3 247 971.9±14 878.3
179.035 500 6.95 [M-H]- 180.042 776 -2.9 96 644.4±1 979.3
3-吲哚甲醛
1H-indole-3-carboxaldehyde		 144.047 447 9.23 [M-H]- 145.054 723 -13.5 781 825.7±31 273.0
赤霉素A4 Gibberellin A4 663.315 967 11.20 [2M-H]- 332.161 622 2.3 1 681 786.4±6 534.3
331.154 800 11.20 [M-H]- 332.162 076 0.9 872 433.9±26 173.0
995.475 686 11.20 [3M-H]- 332.160 987 4.2 5 614.1±130.4
377.159 456 11.20 [M+FA-H]- 332.161 255 3.4 66 434.1±3 986.0
367.130 460 11.20 [M+Cl]- 332.161 058 4.0 80 332.5±312.1
棉酚Gossypol 499.173 942 14.39 [M-H2O-H]- 518.192 332 3.4 34 406.0±3 096.5
517.184 256 14.69 [M-H]- 518.191 532 4.9 322 041.7±7 471.0
赤霉素A3 Gibberellin A3 691.273 483 8.52 [2M-H]- 346.140 380 3.6 1 338 600.7±56 221.2
345.133 419 8.52 [M-H]- 346.140 695 2.7 936 088.3±40 591.3
1 037.412 910 8.52 [3M-H]- 346.140 062 4.6 12 650.3±404.1
381.109 261 8.52 [M+Cl]- 346.139 859 5.1 85 687.1±3 427.5
391.138 037 8.52 [M+FA-H]- 346.139 836 5.2 41 285.3±1 416.3
正离子模式下,检测到的14个标准品中共观察到6种加合物[M+H]+、[M+Na]+、[M+NH4]+、[2M+NH4]+、[2M+Na]+和[2M+H]+。所有标准品都能观察到加合物[M+H]+,白麻苷、绿原酸、蜜二糖、脱落酸、紫云英苷和蔗糖6个标准品观察到[M+Na]+,赤霉素A3、绿原酸、脱落酸、蔗糖和蜜二糖5个标准品观察到[M+NH4]+,青榆烯C和脱落酸均能观察到[2M+Na]+和[2M+NH4]+,仅在脱落酸观察到[2M+H]+。
在负离子模式下,检测到的14个标准品观察到[M-H]-、[2M-H]-、[M+Cl]-、[M+FA-H]-、[3M-H]-、[M+Na-2H]-、[M-H2O-H]-、[M+TFA-H]-8种加合物。所有标准品都能观察到加合物[M-H]-,赤霉素A3、柽柳黄素、赤霉素A4、白麻苷、绿原酸、脱落酸和紫云英苷7个标准品能观察到[2M-H]-,赤霉素A3、赤霉素A4、白麻苷、脱落酸4个标准品能观察到[M+Cl]-,赤霉素A3、赤霉素A4、蔗糖、蜜二糖4个标准品能观察到[M+FA-H]-,赤霉素A3和赤霉素A4均能观察到[3M-H]-,仅在绿原酸中能观察到[M+Na-2H]-,棉酚中能观察到[M-H2O-H]-,白麻苷中能观察到[M+TFA-H]-。

2.4 离子模式对峰强度的影响

基于流动相甲酸乙腈的梯度,特定标准品在UPLC-ESI-MS的正离子模式或负离子模式下有不同的优势。本研究在2种模式下依次分析了棉花代谢产物标准品,以峰强度表示加合物绝对信号强度,其中离子信号最强的为主导加合物。对2个离子模式下各标准品主导加合物的信号进行比较分析,结果表明蜜二糖正离子模式（加合物[M+NH4]+）信号强于负离子模式（[M+FA-H]-）5.4倍,棉酚在2种模式下加合物（[M+H]+、[M-H]-）信号相当,其他12种化合物均是负离子模式信号强于正离子模式,信号强度增加0.5~216倍。这一结果表明,负离子模式可能是棉花次生代谢产物UPLC-ESI-MS分析的最佳检测模式。
图2 棉花代谢产物标准品正离子模式和负离子模式下最强信号离子的峰强度比值对数

Fig. 2 Peak intensity ratios, in logarithmic scale, of the strongest signal ion (peak intensity) obtained in positive and negative ion modes for some cotton metabolite standards

Full size|PPT slide

3 讨论

3.1 代谢组分析中化合物鉴定的推定方法

本研究在对棉花次生代谢产物标准品进行非靶标代谢组质谱分析时,每个标准品的质谱数据包含1 000多个离子数据（表1）,在此数据中发现和鉴定出相应的物质存在很大难度。因此,实现代谢产物高通量准确鉴定一直是重要的课题。Moco等[21]采用HPLC-ESI-MS分析番茄（Solanum lycopersicum）代谢组,并利用番茄次生代谢产物加合物m/z得到计算的准确相对分子质量及其与标准品准确相对分子质量间误差,建立了番茄次生代谢物质的鉴定平台MoTo DB,实现了数据共享。但这一数据库仅限于单个离子数据的搜索,效率较低。Benton等[22]利用在线XCMS软件处理质谱数据,实现质谱数据在METLIN数据库中自动搜索,实现代谢产物高通量鉴定。然而该方案使用的METLIN数据库,偏重于化学分析,虽然含有海量的物质种类,但大多数化合物缺乏与植物代谢产物的紧密关联,在进行植物代谢产物鉴定方面存在较大的局限性。因此,针对具体植物的代谢产物鉴定,仍需建立相应植物代谢产物数据库及其鉴定计算方法。本研究利用MATLAB软件编写的2个用于棉花次生代谢产物鉴定的计算程序POSid和NEGtid,在计算原理上与上述2种方法相同,能够从大量质谱数据中高效、准确发现靶标物质,完成无靶标棉花次生代谢产物的鉴定工作,实现了棉花次生代谢产物快速鉴定。另外,该方法通过UPLC-ESI-MS分析→XCMS质谱数据处理→质谱数据→POSid和NEGtid计算和比较分析等主要步骤完成,整个实施过程均能分割独立完成,方便研究人员自主应用。因此,该方法的建立,为进一步进行棉花次生代谢组分析中代谢产物的准确鉴定提供了高效和便捷的方法支持。迄今为止,已经明确从棉花根、茎、叶、花和种子提取物中分离和检测到的代谢产物111种,包括萜类化合物61种、酚类化合物6种、黄酮类化合物26种、生物碱6种、脂肪族天然产物6种、碳水化合物5种和简单芳香族天然产物1种[23,24]。本研究仅涉及18个棉花代谢产物,继续完善棉花代谢产物数据库信息以及进一步开展棉花次生代谢组研究是棉花生物学基础研究的重点工作之一。

3.2 棉花代谢产物加合物形成

在LC-ESI-MS分析中,加合物形成是对分析物进行定性和定量的基础。明确每个物质在特定LC和ESI条件下形成的加合物,特别是主导加合物,对该物质的LC-ESI-MS分析鉴定尤为重要。Ghosh等[9]在ESI模式下通过甲酸加合物实现了酰基蔗糖代谢物的测定,Mathis等[10]采用负离子加合物建立了高能炸药的定性和定量的方法,Antonowicz等[25]从4种未修饰的核苷中分离出7种候选核苷加合物,建立了1种仅需6 min的超高效液相色谱法,用于小鼠组织样品中羰基、氯和氧加合物的定量分析。所有这些研究表明,在LC-ESI-MS分析中,加合物形成对待鉴定的分析物定性和定量分析具有重要作用。本研究对棉花次生代谢产物标准品在UPLC-ESI-MS分析中加合物形成的加合物种类进行了分析,明确了特定液相色谱和电喷雾离子条件下被鉴定化合物的加合物形成的种类和主导加合物,可为棉花代谢产物的定性和定量分析提供理论数据。

3.3 UPLC-ESI-MS检测时检测模式选择

LC的流动相成分决定了分析物的加合物种类不同,进一步导致ESI正或负离子模式下检测到的信号强度会有较大的差异[26]。Moco等[21]在采用HPLC-ESI-TOF/MS分析番茄代谢物时,发现基于乙腈与甲酸酸化梯度的特定液相色谱流动相,特定化合物在电喷雾正或负离子模式下存在不同加合物信号强度（峰强度）差异,具有ESI模式的选择性。本研究证明供试棉花代谢产物标准品在基于乙腈与甲酸酸化梯度特定液相色谱流动相条件下主导加合物（拥有最大峰强度）信号强度也存在同样规律,具有电喷雾离子模式选择性。如：蜜二糖正离子模式（加合物[M+NH4]+）信号强于负离子模式（[M+FA-H]-）2.7倍,适合正离子模式检测;棉酚在2种模式下加合物（[M+H]-、[M+H]-）信号相当,2种ESI模式均可检测;其他12种标准品均是负离子模式信号强度高出正离子模式0.5~216倍,适合采用负离子模式检测。

4 结论

选用18个棉花代谢产物标准品为供试化合物,建立的基于计算准确分子量的棉花次生代谢产物鉴定MATLAB程序,可实现无靶标质谱数据的快速鉴定。在本研究规定的UPLC和ESI条件下,2种ESI模式下14个化合物得到鉴定,正离子模式下出现6种加合物,负离子模式下出现8种加合物,单个化合物的质谱可观察到1~6种不同的加合物,其中1个为主导加合物。主导加合物具有ESI检测模式的偏好性,决定了该化合物适宜的电喷雾离子模式。这些结果为进一步开展棉花次生代谢组分析研究提供了技术和理论数据支撑。

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基金

国家重点研发计划(2017YFD0201900)
国家现代农业产业技术体系——棉花产业技术体系(CARS-15-18)

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