液相等电聚焦技术结合液相色谱-质谱联用技术分析鼠肝全蛋白

doi:10.7503/cjcu20141120

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (6): 1074.doi: 10.7503/cjcu20141120

液相等电聚焦技术结合液相色谱-质谱联用技术分析鼠肝全蛋白

李颖¹, 姚鋆¹, 杨芃原^1,², 樊惠芝¹()

1. 复旦大学化学系, 上海 200433
2. 复旦大学生物医学研究院, 上海 200032

收稿日期:2014-12-22 出版日期:2015-06-10 发布日期:2015-05-05
作者简介:联系人简介: 樊惠芝, 女, 博士, 教授, 博士生导师, 主要从事蛋白质翻译后乙酰化修饰方面的研究. E-mail:hzfan@fudan.edu.cn
基金资助:
国家重大科学仪器设备开发专项(批准号: 2011YQ030139)、国家自然科学基金(批准号: 20675020)和国家“九七三”计划项目(批准号: 2010CB912704)资助

Global Survey of Mouse Liver Protein Expression Using Liquid Isoelectric Focusing Prefractionation of Tryptic Peptides and LC-MS/MS^†

LI Ying¹, YAO Jun¹, YANG Pengyuan^1,², FAN Huizhi^1,^*()

1. Department of Chemistry, Fudan University, Shanghai 200433, China
2. Institute of Biomedical Sciences, Fudan University, Shanghai 200032, China

Received:2014-12-22 Online:2015-06-10 Published:2015-05-05
Contact: FAN Huizhi E-mail:hzfan@fudan.edu.cn
Supported by:
† Supported by the National Key Program of Scientific Instrument Development of China(No.2011YQ030139), the National Natural Science Foundation of China(No.20675020) and the National Basic Research Program of China(No.2010CB912704)

摘要/Abstract

摘要：

利用液相等电聚焦预分离技术结合液相色谱-质谱(LTQ-Orbitrap)联用技术, 研究了C57小鼠肝脏的蛋白质表达谱. 质谱分析结果采用MaxQuant1.4.1.2软件搜索数据库, 共鉴定出3474个蛋白(2个以上唯一肽段). 用DAVID在线工具、 GO分类工具和IPA软件对鉴定蛋白进行生物信息学分析, 单独发现832个新蛋白. 研究结果表明, 通过基于等电点和亲疏水性的两维分离, 提高了质谱鉴定蛋白数, 可以鉴定出更多低丰度蛋白.

关键词: 蛋白质组学, 液相等电聚焦, 液相色谱-质谱联用, 鼠肝蛋白

Abstract:

To comprehensively and quickly identify proteins of mouse liver, the tryptic peptides were pre-fractioned and enriched by isoelectric focusing technigue using Agilent 3100 OFFGEL Fractionator into 24 fractions, followed by RPLC and MS/MS analysis. Each MS/MS spectrum was searched for mouse species against the Swissprot database. The results were analyzed which resulted in the identification of proteins in mouse liver. Finally, 3474 proteins were identified from mouse liver, 832 of them were new identified proteins. Bioinformatic analyses were performed to demonstrate the physicochemical properties, cellular locations and functions of the proteins. The combined use of liquid fractionation and LC-MS allows a considerable increase in proteome coverage of very complex samples prepared from the tissues extracts and supports the low-concentrated protein identification. This study can provide useful data to research C57 mouse liver proteins.

Key words: Proteomics, Liquid isoelectric focusing, LC-MS/MS, Mouse liver protein

中图分类号:

O657

TrendMD:

李颖, 姚鋆, 杨芃原, 樊惠芝. 液相等电聚焦技术结合液相色谱-质谱联用技术分析鼠肝全蛋白. 高等学校化学学报, 2015, 36(6): 1074.

LI Ying, YAO Jun, YANG Pengyuan, FAN Huizhi. Global Survey of Mouse Liver Protein Expression Using Liquid Isoelectric Focusing Prefractionation of Tryptic Peptides and LC-MS/MS^†. Chem. J. Chinese Universities, 2015, 36(6): 1074.

图/表 9

Fig.1 Analysis workflow used to profile mouse liver proteins

Fig.2 Number distribution of the identified peptides in OFFGEL fraction wells(A) and fraction distribution of the identified proteins(B)

Fig.3 Cellular components GO mapping of the identified proteins(A) and biological process GO mapping of the identified proteins(B)

Table 1 Number of protein identification classified by number of peptides used for identifications

Protein number	Protein number of four or more unique peptide	Protein number of three unique peptide	Protein number of two unique peptide
3474	2433(70%)	378(11%)	663(19%)
7099^[20]	2857(40%)^[20]	1475(21%)^[20]	2758(39%)^[20]

Fig.4 Comparison of four mouse liver proteomes

Table 2 Distribution of abundance of proteins(iBAQ intensities) identified with FDR of 1%

Protein number	Number of iBAQ intensities 1.0×10⁹—1.0×10⁷ of protein	Number of iBAQ intensities 1.0×10⁷—1.0×10⁵ of protein	Number of iBAQ intensities 1.0×10⁵—1.0×10³ of protein
3474^a	470(13.5%)	2312(66.5%)	692(20%)
832^b	8(1%)	527(63%)	299(36%)

Fig.5 Cellular components GO mapping of new identified proteins(A) and KEGG pathways mapping of new identified proteins(B)

Fig.6 Distribution of peptides of new proteins identified in relation to their theoretical molecular mass(A), pI(B) and GRAVY values(C)(A) Molecular weight: a. 700—1000; b. 1100—1500; c. 1500—1900; d. 1900—2300; e. 2300—2700; f. 2700—3100; g. >3100. (B) pI: a. 3—4; b. 4—5; c. 5—6; d. 6—7; e. 7—8; f. 8—9; g. 9—10; h. 10—11; i. 11—12; j. 12—13; (C) GRAVY value: a. >0; b. -0.25—0; c. -0.5—0.25; d. -0.75—-0.5; e. -1.00—-0.75; f. <-1.00.

Fig.7 TIC of peptide fraction eluted(A) and MS spectrum(inset) of peptides effluent(at 108.2 min) labled by ★ in (A) and MS/MS spectrum(B) for precursor ion of m/z 685.20 labled by ★ in the inset of (A)

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液相等电聚焦技术结合液相色谱-质谱联用技术分析鼠肝全蛋白

Global Survey of Mouse Liver Protein Expression Using Liquid Isoelectric Focusing Prefractionation of Tryptic Peptides and LC-MS/MS^†

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液相等电聚焦技术结合液相色谱-质谱联用技术分析鼠肝全蛋白

Global Survey of Mouse Liver Protein Expression Using Liquid Isoelectric Focusing Prefractionation of Tryptic Peptides and LC-MS/MS†

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Global Survey of Mouse Liver Protein Expression Using Liquid Isoelectric Focusing Prefractionation of Tryptic Peptides and LC-MS/MS^†