基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展

doi:10.12211/2096-8280.2023-031

合成生物学 ›› 2023, Vol. 4 ›› Issue (5): 1000-1019.DOI: 10.12211/2096-8280.2023-031

基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展

吴玉洁¹^,²^,³, 刘欣欣¹, 刘健慧¹, 杨开广¹, 随志刚¹, 张丽华¹, 张玉奎¹

^1.中国科学院大连化学物理研究所，中国科学院分离分析化学重点实验室，辽宁大连 116023
^2.大连理工大学化学学院，辽宁大连 116024
^3.中国科学院大学，北京 100049

收稿日期:2023-04-17 修回日期:2023-06-26 出版日期:2023-10-31 发布日期:2023-11-15
通讯作者: 杨开广,随志刚
作者简介:吴玉洁（1998—），女，博士研究生。研究方向为面向合成生物学的蛋白质组学分析方法。E-mail：yujiewu@dicp.ac.cn
杨开广（1981—），男，研究员，博士生导师。研究方向为蛋白质组学新技术新方法。E-mail：yangkaiguang@dicp.ac.cn
随志刚（1979—），男，副研究员，硕士生导师。研究方向为基于质谱的蛋白质及代谢物高通量分析方法开发。E-mail：zhigangsui@dicp.ac.cn
基金资助:
国家自然科学基金(22104138);中国科学院“青年创新促进会”优秀会员基金(Y2021058)

Research progress of strain screening and quantitative analysis of key molecules based on high-throughput liquid chromatography and mass spectrometry

Yujie WU¹^,²^,³, Xinxin LIU¹, Jianhui LIU¹, Kaiguang Yang¹, Zhigang SUI¹, Lihua ZHANG¹, Yukui ZHANG¹

^1.Key Laboratory of Separation Science for Analytical Chemistry，Dalian Institute of Chemical Physics，Chinese Academy of Sciences，Dalian 116023，Liaoning，China
^2.School of Chemistry，Dalian University of Technology，Dalian 116024，Liaoning，China
^3.University of Chinese Academy of Sciences，Beijing 100049，China

Received:2023-04-17 Revised:2023-06-26 Online:2023-10-31 Published:2023-11-15
Contact: Kaiguang Yang, Zhigang SUI

摘要/Abstract

摘要：

合成生物学是21世纪新兴的交叉学科，已经为医药、化工、能源、食品和农业等领域带来了显著的改变。微生物细胞工厂（microbial cell factory，MCF）的构建和应用是合成生物学的重要研究内容，正迅速向实用化、产业化的方向发展。得益于基因编辑技术的进步，MCF菌株文库构建的能力大幅提升，亦产生了数量庞大的待测样本，亟需发展高通量自动化的分析检测方法与之匹配。此外，针对关键代谢酶或代谢物的高通量检测技术，也对MCF改造乃至工业化生产有重要的指导意义。液相色谱质谱技术是生物分子检测分析的重要手段，在蛋白质、代谢物定量分析方面占据重要地位。因此本文总结回顾了近年来高通量液相色谱质谱技术在微生物菌株筛选及关键分子定量分析方面的研究进展，从样品制备、色谱分离、质谱分析及数据处理等层面展开阐述，并对这一领域与自动化平台结合的前景及如何深度融合的挑战进行简要概括，为推动基于合成生物学生物智造领域的快速发展提供指导。

关键词: 液相色谱, 质谱, 高通量筛选, 蛋白质组学, 代谢组学, 合成生物学, 微生物细胞工厂

Abstract:

Synthetic biology is an emerging interdisciplinary research area and has brought significant changes to fields such as medicine, chemical engineering, energy, food and agriculture. The construction and application of microbial cell factories (MCFs) is an important task of synthetic biology, which will lead the way to a sustainable industrial-scale manufacturing sector. With the progress of genome editing technology, the construction strategy of MCFs has evolved from random mutation to customized transformation at the whole genome level. Biologists can obtain 10³~10⁷ strain mutant libraries in a short time, creating an urgent need for the development of high-throughput screening methods. In addition, the demand for precise quantitative analysis of key molecules in metabolic pathways, such as metabolic enzymes and metabolites, is increasingly prominent in the process of strain transformation, evolution, and fermentation monitoring. Liquid chromatography (LC) offers excellent separation capability, allowing efficient separation of target molecules, while mass spectrometry (MS) provides strong detection ability due to its high specificity and sensitivity. Therefore, LC and MS techniques have been widely applied in the field of life sciences, especially for the qualitative and quantitative analyses of proteins and metabolites. With the development of high-throughput LC and MS technologies, these methods have become powerful tools for screening strains and quantitative research of key molecules in synthetic biology. Therefore, this work reviews the research progresses of LC and/or MS based strain screening and quantification of key molecules, focusing on the following aspects: sample preparation, chromatographic separation, mass spectrometric analysis and data processing. Finally, we briefly summarize the future prospects and challenges in this field. It is expected that with the continuous progress of LC and MS, the deep integration with automatic devices and data processing platforms, the advantages of LC and MS in high-throughput screening of synthetic biology strains and quantitative analysis of key molecules will become increasingly prominent, which will certainly promote the rapid development of biological intelligence based on synthetic biology.

Key words: liquid chromatography, mass spectrometry, high-throughput screening, proteomics, metabolomics, synthetic biology, microbial cell factory

中图分类号:

O657.63

吴玉洁, 刘欣欣, 刘健慧, 杨开广, 随志刚, 张丽华, 张玉奎. 基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展[J]. 合成生物学, 2023, 4(5): 1000-1019.

Yujie WU, Xinxin LIU, Jianhui LIU, Kaiguang Yang, Zhigang SUI, Lihua ZHANG, Yukui ZHANG. Research progress of strain screening and quantitative analysis of key molecules based on high-throughput liquid chromatography and mass spectrometry[J]. Synthetic Biology Journal, 2023, 4(5): 1000-1019.

图/表 8

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质谱技术	优缺点	菌株培养方式	筛选通量	适用对象	应用示例	参考文献
MALDI	优点：高耐盐性，易于操作，样品用量少，分析速度快，通量高缺点：需要添加基质，可能引入基质峰干扰	琼脂平板	约2 s/样品	可用于分析脂肪酸等小分子及蛋白质等生物大分子	通过检测酰基链磷脂酰胆碱，以筛选高产中链脂肪酸的酿酒酵母菌株	[45]
		琼脂平板	<2.5 s/样品		机器视觉算法辅助识别随机分布菌落，应用于大肠杆菌天然产物文库筛选	[22]
		琼脂平板/ 微孔板	<5 s/样品		开发了菌落位置转换MALDI坐标算法，用于酿酒酵母、大肠杆菌及荧光假单胞菌文库筛选	[46]
		微孔板	约5 s/样品		筛选大肠杆菌环二肽合酶突变体文库及羊毛硫肽化合物文库，基本实现整个流程自动化	[47-48]
		液滴	约5 s/样品		酵母甜蛋白及植酸酶产物文库筛选	[50-51]
SAMDI	优点：特异性强，可用于检测复杂体系中的目标分子缺点：无法区分分子量相同的产物和底物	琼脂平板	<1 s/样品	可用于分析小分子	利用自组转单层技术在靶板上修饰马来酰亚胺基团，筛选大肠杆菌细胞色素P411突变体文库	[23]
NIMS	优点：灵敏度高，无需添加基质缺点：需要构建纳米结构表面	琼脂平板	<1 s/样品	可用于分析小分子、脂质和肽	结合点击化学，筛选大肠杆菌细胞色素P450突变体文库	[25]
ESI	优点：采用软电离方式，基本不产生碎片峰，分辨率高缺点：耐盐性较差，取样效率低	微孔板	约84 s/样品	可用于分析极性强、热稳定性差的小分子和生物大分子	液相色谱质谱联用快速筛选三萜桦木酸产量提高的酿酒酵母菌株	[26]
		液滴	约1 s/样品		在线筛选谷氨酸棒杆菌文库	[36]
		微孔板	约60 s/样品		融合LESA技术，用于酵母菌株衣康酸、三乙酸内酯和棕榈酸产物定量筛选分析	[61]
DESI	优点：高耐盐性，可实现原位实时分析，通量高缺点：电喷雾溶剂组成可能影响样品溶解与电离	琼脂平板	<1 s/样品	可用于分析非极性小分子及极性大分子	与成像技术结合，快速筛选大肠杆菌突变体文库	[64-65]
LA-REI	优点：易于操作，无需样品制备，快速分析缺点：无法区分异构体	琼脂平板	约10 s/样品	可用于分析小分子代谢物	激光辅助实现酵母白桦酸产物文库筛选	[67]

质谱技术	优缺点	菌株培养方式	筛选通量	适用对象	应用示例	参考文献
MALDI	优点：高耐盐性，易于操作，样品用量少，分析速度快，通量高缺点：需要添加基质，可能引入基质峰干扰	琼脂平板	约2 s/样品	可用于分析脂肪酸等小分子及蛋白质等生物大分子	通过检测酰基链磷脂酰胆碱，以筛选高产中链脂肪酸的酿酒酵母菌株	[45]
		琼脂平板	<2.5 s/样品		机器视觉算法辅助识别随机分布菌落，应用于大肠杆菌天然产物文库筛选	[22]
		琼脂平板/ 微孔板	<5 s/样品		开发了菌落位置转换MALDI坐标算法，用于酿酒酵母、大肠杆菌及荧光假单胞菌文库筛选	[46]
		微孔板	约5 s/样品		筛选大肠杆菌环二肽合酶突变体文库及羊毛硫肽化合物文库，基本实现整个流程自动化	[47-48]
		液滴	约5 s/样品		酵母甜蛋白及植酸酶产物文库筛选	[50-51]
SAMDI	优点：特异性强，可用于检测复杂体系中的目标分子缺点：无法区分分子量相同的产物和底物	琼脂平板	<1 s/样品	可用于分析小分子	利用自组转单层技术在靶板上修饰马来酰亚胺基团，筛选大肠杆菌细胞色素P411突变体文库	[23]
NIMS	优点：灵敏度高，无需添加基质缺点：需要构建纳米结构表面	琼脂平板	<1 s/样品	可用于分析小分子、脂质和肽	结合点击化学，筛选大肠杆菌细胞色素P450突变体文库	[25]
ESI	优点：采用软电离方式，基本不产生碎片峰，分辨率高缺点：耐盐性较差，取样效率低	微孔板	约84 s/样品	可用于分析极性强、热稳定性差的小分子和生物大分子	液相色谱质谱联用快速筛选三萜桦木酸产量提高的酿酒酵母菌株	[26]
		液滴	约1 s/样品		在线筛选谷氨酸棒杆菌文库	[36]
		微孔板	约60 s/样品		融合LESA技术，用于酵母菌株衣康酸、三乙酸内酯和棕榈酸产物定量筛选分析	[61]
DESI	优点：高耐盐性，可实现原位实时分析，通量高缺点：电喷雾溶剂组成可能影响样品溶解与电离	琼脂平板	<1 s/样品	可用于分析非极性小分子及极性大分子	与成像技术结合，快速筛选大肠杆菌突变体文库	[64-65]
LA-REI	优点：易于操作，无需样品制备，快速分析缺点：无法区分异构体	琼脂平板	约10 s/样品	可用于分析小分子代谢物	激光辅助实现酵母白桦酸产物文库筛选	[67]

质谱采集类型	菌株样本	分析通量（每天检测样本数估值）	参考文献
靶向采集	大肠杆菌E.coil	25 min分离梯度定量大肠杆菌中600多条肽（约50样品/天）	[96]
非靶向采集	酿酒酵母S.cerevistae	19 min有效分离梯度定量检测796个酵母菌株蛋白质组（约70样品/天）	[97]
非靶向采集	大肠杆菌E.coil	4 min分离梯度定量226个脂类分子（约320样品/天）	[99]
非靶向采集	酿酒酵母S.cerevistae	15 min分离梯度结合双分析柱系统检测蛋白（约90样品/天）	[104]
非靶向采集	酿酒酵母S.cerevistae	23 min分离梯度定量检测100个酵母蛋白质组（约45样品/天）	[115]
非靶向采集	酿酒酵母S.cerevistae	5 min分离梯度定量1900种蛋白（约280样品/天）	[116]
靶向采集	大肠杆菌E.coil	10 min分离梯度定量400多种蛋白质（约130样品/天）	[124]
靶向采集	恶臭假单胞菌P.putida KT2440	32 min分离梯度定量132种蛋白质（约40样品/天）	[125]
靶向采集	大肠杆菌E.coil	5 min分离梯度检测102种代谢物（约280样品/天）	[126]

质谱采集类型	菌株样本	分析通量（每天检测样本数估值）	参考文献
靶向采集	大肠杆菌E.coil	25 min分离梯度定量大肠杆菌中600多条肽（约50样品/天）	[96]
非靶向采集	酿酒酵母S.cerevistae	19 min有效分离梯度定量检测796个酵母菌株蛋白质组（约70样品/天）	[97]
非靶向采集	大肠杆菌E.coil	4 min分离梯度定量226个脂类分子（约320样品/天）	[99]
非靶向采集	酿酒酵母S.cerevistae	15 min分离梯度结合双分析柱系统检测蛋白（约90样品/天）	[104]
非靶向采集	酿酒酵母S.cerevistae	23 min分离梯度定量检测100个酵母蛋白质组（约45样品/天）	[115]
非靶向采集	酿酒酵母S.cerevistae	5 min分离梯度定量1900种蛋白（约280样品/天）	[116]
靶向采集	大肠杆菌E.coil	10 min分离梯度定量400多种蛋白质（约130样品/天）	[124]
靶向采集	恶臭假单胞菌P.putida KT2440	32 min分离梯度定量132种蛋白质（约40样品/天）	[125]
靶向采集	大肠杆菌E.coil	5 min分离梯度检测102种代谢物（约280样品/天）	[126]

软件工具	分析对象	网址	参考文献
AB3D	蛋白质组	http://www.first-ms3d.jp/english/（Mass++插件）	[132]
DynaMet	代谢组	https://pypi.python.org/pypi/dynamet/	[137]
DIA-NN	蛋白质组	https://github.com/vdemichev/diann	[140]
Tric	蛋白质组	http://proteomics.ethz.ch/tric/	[141]
MRMAnalyzer	代谢组	http://link.springer.com/10.1007/s11306-015-0809-4（R包）	[142]
MESSI	代谢组	http://sbb.hku.hk/MESSI/	[144]
PTools	代谢组	https://brg.ai.sri.com/ptools/	[145]

基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展

Research progress of strain screening and quantitative analysis of key molecules based on high-throughput liquid chromatography and mass spectrometry

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