创新数据非依赖性采集用于复杂基质目标蛋白质的定量分析

张等伟
摘 要 数据非依赖性采集(DIA)是随着定量蛋白质组学而建立的质谱扫描技术。DIA能够获得扫描范围内所有母离子及二级子离子信息,不会造成低丰度离子信息的丢失,同时突破了高分辨质谱二级定量的通量限制。本研究基于静电场轨道阱QqITOT三合一质谱,发展了经典DIA方法以及WiSIMDIA和Full MSDIA两种全新DIA方法,并对Hela细胞全蛋白中添加的10条低浓度肽段进行定量分析,考察方法的线性、重现性和灵敏度。结果表明,3种方法的定量限均低至amol (14~435 amol),并展示出良好的线性和定性确证可靠性。其中,WiSIMDIA基于超高分辨一级监测定量,与经典DIA优势互补;Full MSDIA的选择窗口仅3 amu,能够直接进行搜库鉴定,实现了数据依赖性采集(DDA)和DIA的统一,摆脱了DIA依赖于DDA建立谱图库的局限性。
1 引 言
数据依赖性采集(Data dependent acquisition, DDA)是串联质谱非目标化合物分析的主要手段。蛋白质组学的经典策略鸟枪法(Shotgun)即基于DDA发展而来,利用一级全扫描检测肽段母离子,然后按信号强度排列,将前若干位的母离子依次选择、碎裂,并扫描二级碎片离子。同时,动态排除、价态排除、中性丢失/诊断离子触发等技术,使DDA尽可能多地采集有效肽段谱图,实现鉴定结果最大化[1]。基于鸟枪法,蛋白质组学已经实现酵母蛋白质组接近完全覆盖,人类蛋白质组也已达到50%以上的基因组覆盖和7个数量级的动态范围[2,3]。然而,DDA的局限性也逐渐显现:(1) 先强后弱的采集方式易造成低丰度肽段信息丢失;(2) 母离子选择有一定的随机性,造成重现性不佳;(3) 每个循环获得的谱图数量不一,造成扫描点数不均匀,影响定量分析准确性。
目标蛋白质组学针对目标蛋白/肽段离子实时监测和采集,避免了DDA的信息丢失和重现性问题。主要采集方法包括选择离子监测(Selected ion monitoring, SIM)、基于三重四极杆的选择反应监测(Selected reaction monitoring, SRM)和基于高分辨质谱的平行反应监测(Parallel reaction monitoring, PRM),是目标蛋白验证和绝对定量的有效手段[4,5]。但是目标性的采集方法需要指定目标肽段,对于未知肽段无法采集;通量限制也使得一次实验只能监测数量有限肽段或离子对,难以满足大规模蛋白分析的需要。
数据非依赖性采集(Data independent acquisition, DIA)使用25 amu或更大间隔将整个质量范围等分为若干窗口,每个窗口依次选择、碎裂、扫描。DIA能够获得质量范围内所有母离子的全部碎片离子信息,通量无上限,循环时间固定,同时数据可以回溯,有效解决了DDA和目标采集方法存在的问题[6]。目前,已发展了多种基于飞行时间(QTOF)、静电场轨道阱(Orbitrap)和离子阱的DIA方法[7]。Gillet等使用32个连续的25 amu窗口,基于QTOF (TripleTOF 5600)发展了SWATH技术,并证明该技术的定量能力与SRM相当[8]。Egertson等利用QOrbitrap (Q Exactive)独有的多重扫描功能(Multiplexing, MSX)发展了MSXDIA技术,将选择窗口缩小到4 amu,最大程度减少了共流出肽段和杂质的干扰[9]。
然而,传统数据非依赖性采集仍存在诸多局限:(1) 由于扫描速度的限制,DIA难以使用超高分辨率扫描;(2) DIA的大窗口选择引入较大干扰,虽然MSXDIA缩小了选择窗口,但需要特定的算法解析数据,增加了工作量;(3) DIA依赖于DDA建立的谱图库进行匹配,实现定性确证和定量离子选择,因此DDA鉴定不到的蛋白,DIA也无法分析。
本研究基于四极杆静电场轨道阱线性离子阱(QOTqIT)三合一质谱,利用添加10条低浓度肽段的Hela样本,发展并考察了3种数据非依赖性采集方法,包括经典的DIA、全新的宽窗口SIM扫描DIA (WiSIMDIA)和全扫描DIA (Full MSDIA),定量限均达到amol水平,线性、重现性良好。其中,WiSIMDIA和Full MSDIA基于24万超高分辨率,利用一级精确质量数定量、二级离子阱定性确证,进一步缩小了选择窗口,提高了检测特异性。此外,FullMS DIA可以直接搜库,实现了DDA与DIA的统一,蛋白鉴定数量与DDA相当,摆脱了谱图库的限制。基于QOTqIT的数据非依赖性采集方法灵活多样、流程简单有效,在目标蛋白质组学领域具有广阔的应用前景。
2 实验部分
2.1 仪器与试剂
Orbitrap Fusion三合一质谱仪、EASYnLC 1000纳流超高效液相色谱(Thermo Fisher Scientific)。标准肽段由生工生物(上海)股份有限公司合成;乙腈、甲酸(Thermo Fisher Scientific);其它试剂均购自SigmaAldrich公司。
2.2 Hela细胞全蛋白酶解液制备
出良好的线性、重现性和灵敏度。3种方法的最低定量限均达到amol级,其中肽段GEEMEEMVQSAR
在DIA中最低定量限达14 amol,超越了常规SRM和PRM的定量水平。比较3种方法可以看出,DIA与WiSIMDIA结果差异不大,证明了基于高分辨的二级定量和基于超高分辨的一级定量选择性相当,均能有效排除基质和共流出肽段的干扰,定量能力出色。两种方法又各有特点,形成优势互补:DIA通过四极杆和Orbitrap两级筛选,能有效分析极复杂的样品;WiSIMDIA使用母离子定量,避免了碎裂过程中的损失,在相对简单的基质中灵敏度更高。
3.4 直接搜库鉴定的考察与比较
由于选择窗口过大,同时二级谱图无法与一级母离子相关联,传统数据非依赖性采集无法直接搜库,需要谱图库匹配才能进行定性确证,使DIA的应用受制于DDA。
Full MSDIA基于一级全扫描定量,母离子未经过前级质量分析器选择,因此相比DIA和WiSIMDIA受到的干扰更大,灵敏度略低。但是,Full MSDIA将二级选择窗口缩短到3 amu,与传统DDA的选择窗口相当,能够作为低分辨数据,直接用于数据库检索(相当于母离子质量精度为±1.5 amu),摆脱了谱图库的限制,实现了DDA与DIA的统一。
图4展示了肽段YLGYLEQLLR谱图的直接搜库结果。DDA通常以2 amu为选择窗口,与Full MSDIA 3 amu选择窗口相差不大。搜库时,Full MSDIA一级质量精度以窗口宽度为限,即±1.5 amu,类似于低分辨质谱DDA数据的搜库鉴定。结果显示,Full MSDIA与DDA的二级谱图高度相似,虽然Full MSDIA谱图等同于低分辨数据,但鉴定结果没有明显差异,序列匹配完全一致。
4 结 论
基于静电场轨道阱QqITOT质谱建立DIA、WiSIMDIA、Full MSDIA 3种数据非依赖性采集方法,并使用添加10条低浓度肽段的Hela细胞全蛋白样本对方法进行考察。结果表明,3种方法的定量限均在14~435 amol范围内,线性关系与重现性良好,定性确证可靠性高。其中,WiSIMDIA基于超高分辨SIM定量,与经典DIA二级定量具有一定的互补性;而Full MSDIA将二级选择窗口缩短到3 amu,实现了DIA数据直接搜库鉴定,共从100 ng Hela细胞全蛋白样本鉴定到2835个非冗余蛋白,与DDA鉴定结果重合度高。基于QqITOT的创新数据非依赖性采集方法为定量蛋白质组学提供了全新视角与策略。
References
1 Kalli A, Smith G T, Sweredoski M J, Hess S. J. Proteome Res., 2013, 12(7): 3071-3086
2 Nagaraj N, Kulak N A, Cox J, Neuhauser N, Mayr K, Hoerning O, Vorm O, Mann M. Mol. Cell. Proteomics, 2012, 11(3): M111.013722
3 Geiger T, Wehner A, Schaab C, Cox J, Mann M. Mol. Cell. Proteomics, 2012, 11(3): M111.014050
4 Gallien S, Duriez E, Crone C, Kellmann M, Moehring T, Domon B. Mol. Cell. Proteomics, 2012, 11(12): 1709-1723
5 Gallien S, Duriez E, Demeure K, Domon B. J. Proteomics, 2013, 81: 148-158
6 Chapman J D, Goodlett D R, Masselon C D. Mass Spectrom. Rev., 2013: 10.1002/mas.21400
7 Law K P, Lim Y P. Expert Rev. Proteomics, 2013, 10(6): 551-566
8 Gillet L C, Navarro P, Tate S, Rost H, Selevsek N, Reiter L, Bonner R, Aebersold R. Mol. Cell. Proteomics, 2012, 11(6): O111.016717
9 Egertson J D, Kuehn A, Merrihew G E, Bateman N W, MacLean B X, Ting Y S, Canterbury J D, Marsh D M, Kellmann M, Zabrouskov V, Wu C C, MacCoss M J. Nat. Methods, 2013, 10(8): 744-746
10 Senko M W, Remes P M, Canterbury J D, Mathur R, Song Q, Eliuk S M, Mullen C, Earley L, Hardman M, Blethrow J D, Bui H, Specht A, Lange O, Denisov E, Makarov A, Horning S, Zabrouskov V. Anal. Chem., 2013, 85(24): 11710-11714
Quantification Analysis of Targeted Proteins in Complex
Sample by Novel Data Independent Acquisition
ZHANG Wei1, Reiko Kiyonami2, JIANG Zheng*1, CHEN Wei1
1(Thermo Fisher Scientific, Shanghai 201206, China)
2(Thermo Fisher Scientific, San Jose, CA, USA)
Abstract Data independent acquisition (DIA) is a novel MS scan mode for quantitative proteomics, acquiring all precursors as well as fragments without any loss of low abundant ions, and breaks the throughput limitation of product ion quantification by highresolution MS. Here we developed three DIA methods on quadrupolelinear ion trapOrbitrap (QqITOT) Tribrid MS, classic DIA, as well as novel wide isolation window SIM scan (WiSIM)DIA and full scanDIA (Full MSDIA). Quantitative analysis of 10 low abundant peptides spiked in Hela cell digest was performed by the three methods for linearity, reproducibility and sensitivity evaluation. The results showed that the LOQs reached amol level (14-435 amol) with good linearity and effective MS/MS confirmation. WiSIMDIA utilizes ultrahigh resolution SIM scan for quantification complementary with classic DIA. The isolation window of Full MSDIA was down to 3 amu, and the data could be directly used for database searching, thus realizing the integration of data dependent acquisition (DDA) and DIA, and avoiding the limitation of using spectra library.
Keywords Orbitrap; Data independent acquisition; Proteomics; Absolute quantification
(Received 24 April 2014; accepted 4 July 2014)
由于选择窗口过大,同时二级谱图无法与一级母离子相关联,传统数据非依赖性采集无法直接搜库,需要谱图库匹配才能进行定性确证,使DIA的应用受制于DDA。
Full MSDIA基于一级全扫描定量,母离子未经过前级质量分析器选择,因此相比DIA和WiSIMDIA受到的干扰更大,灵敏度略低。但是,Full MSDIA将二级选择窗口缩短到3 amu,与传统DDA的选择窗口相当,能够作为低分辨数据,直接用于数据库检索(相当于母离子质量精度为±1.5 amu),摆脱了谱图库的限制,实现了DDA与DIA的统一。
图4展示了肽段YLGYLEQLLR谱图的直接搜库结果。DDA通常以2 amu为选择窗口,与Full MSDIA 3 amu选择窗口相差不大。搜库时,Full MSDIA一级质量精度以窗口宽度为限,即±1.5 amu,类似于低分辨质谱DDA数据的搜库鉴定。结果显示,Full MSDIA与DDA的二级谱图高度相似,虽然Full MSDIA谱图等同于低分辨数据,但鉴定结果没有明显差异,序列匹配完全一致。
4 结 论
基于静电场轨道阱QqITOT质谱建立DIA、WiSIMDIA、Full MSDIA 3种数据非依赖性采集方法,并使用添加10条低浓度肽段的Hela细胞全蛋白样本对方法进行考察。结果表明,3种方法的定量限均在14~435 amol范围内,线性关系与重现性良好,定性确证可靠性高。其中,WiSIMDIA基于超高分辨SIM定量,与经典DIA二级定量具有一定的互补性;而Full MSDIA将二级选择窗口缩短到3 amu,实现了DIA数据直接搜库鉴定,共从100 ng Hela细胞全蛋白样本鉴定到2835个非冗余蛋白,与DDA鉴定结果重合度高。基于QqITOT的创新数据非依赖性采集方法为定量蛋白质组学提供了全新视角与策略。
References
1 Kalli A, Smith G T, Sweredoski M J, Hess S. J. Proteome Res., 2013, 12(7): 3071-3086
2 Nagaraj N, Kulak N A, Cox J, Neuhauser N, Mayr K, Hoerning O, Vorm O, Mann M. Mol. Cell. Proteomics, 2012, 11(3): M111.013722
3 Geiger T, Wehner A, Schaab C, Cox J, Mann M. Mol. Cell. Proteomics, 2012, 11(3): M111.014050
4 Gallien S, Duriez E, Crone C, Kellmann M, Moehring T, Domon B. Mol. Cell. Proteomics, 2012, 11(12): 1709-1723
5 Gallien S, Duriez E, Demeure K, Domon B. J. Proteomics, 2013, 81: 148-158
6 Chapman J D, Goodlett D R, Masselon C D. Mass Spectrom. Rev., 2013: 10.1002/mas.21400
7 Law K P, Lim Y P. Expert Rev. Proteomics, 2013, 10(6): 551-566
8 Gillet L C, Navarro P, Tate S, Rost H, Selevsek N, Reiter L, Bonner R, Aebersold R. Mol. Cell. Proteomics, 2012, 11(6): O111.016717
9 Egertson J D, Kuehn A, Merrihew G E, Bateman N W, MacLean B X, Ting Y S, Canterbury J D, Marsh D M, Kellmann M, Zabrouskov V, Wu C C, MacCoss M J. Nat. Methods, 2013, 10(8): 744-746
10 Senko M W, Remes P M, Canterbury J D, Mathur R, Song Q, Eliuk S M, Mullen C, Earley L, Hardman M, Blethrow J D, Bui H, Specht A, Lange O, Denisov E, Makarov A, Horning S, Zabrouskov V. Anal. Chem., 2013, 85(24): 11710-11714
Quantification Analysis of Targeted Proteins in Complex
Sample by Novel Data Independent Acquisition
ZHANG Wei1, Reiko Kiyonami2, JIANG Zheng*1, CHEN Wei1
1(Thermo Fisher Scientific, Shanghai 201206, China)
2(Thermo Fisher Scientific, San Jose, CA, USA)
Abstract Data independent acquisition (DIA) is a novel MS scan mode for quantitative proteomics, acquiring all precursors as well as fragments without any loss of low abundant ions, and breaks the throughput limitation of product ion quantification by highresolution MS. Here we developed three DIA methods on quadrupolelinear ion trapOrbitrap (QqITOT) Tribrid MS, classic DIA, as well as novel wide isolation window SIM scan (WiSIM)DIA and full scanDIA (Full MSDIA). Quantitative analysis of 10 low abundant peptides spiked in Hela cell digest was performed by the three methods for linearity, reproducibility and sensitivity evaluation. The results showed that the LOQs reached amol level (14-435 amol) with good linearity and effective MS/MS confirmation. WiSIMDIA utilizes ultrahigh resolution SIM scan for quantification complementary with classic DIA. The isolation window of Full MSDIA was down to 3 amu, and the data could be directly used for database searching, thus realizing the integration of data dependent acquisition (DDA) and DIA, and avoiding the limitation of using spectra library.
Keywords Orbitrap; Data independent acquisition; Proteomics; Absolute quantification
(Received 24 April 2014; accepted 4 July 2014)
由于选择窗口过大,同时二级谱图无法与一级母离子相关联,传统数据非依赖性采集无法直接搜库,需要谱图库匹配才能进行定性确证,使DIA的应用受制于DDA。
Full MSDIA基于一级全扫描定量,母离子未经过前级质量分析器选择,因此相比DIA和WiSIMDIA受到的干扰更大,灵敏度略低。但是,Full MSDIA将二级选择窗口缩短到3 amu,与传统DDA的选择窗口相当,能够作为低分辨数据,直接用于数据库检索(相当于母离子质量精度为±1.5 amu),摆脱了谱图库的限制,实现了DDA与DIA的统一。
图4展示了肽段YLGYLEQLLR谱图的直接搜库结果。DDA通常以2 amu为选择窗口,与Full MSDIA 3 amu选择窗口相差不大。搜库时,Full MSDIA一级质量精度以窗口宽度为限,即±1.5 amu,类似于低分辨质谱DDA数据的搜库鉴定。结果显示,Full MSDIA与DDA的二级谱图高度相似,虽然Full MSDIA谱图等同于低分辨数据,但鉴定结果没有明显差异,序列匹配完全一致。
4 结 论
基于静电场轨道阱QqITOT质谱建立DIA、WiSIMDIA、Full MSDIA 3种数据非依赖性采集方法,并使用添加10条低浓度肽段的Hela细胞全蛋白样本对方法进行考察。结果表明,3种方法的定量限均在14~435 amol范围内,线性关系与重现性良好,定性确证可靠性高。其中,WiSIMDIA基于超高分辨SIM定量,与经典DIA二级定量具有一定的互补性;而Full MSDIA将二级选择窗口缩短到3 amu,实现了DIA数据直接搜库鉴定,共从100 ng Hela细胞全蛋白样本鉴定到2835个非冗余蛋白,与DDA鉴定结果重合度高。基于QqITOT的创新数据非依赖性采集方法为定量蛋白质组学提供了全新视角与策略。
References
1 Kalli A, Smith G T, Sweredoski M J, Hess S. J. Proteome Res., 2013, 12(7): 3071-3086
2 Nagaraj N, Kulak N A, Cox J, Neuhauser N, Mayr K, Hoerning O, Vorm O, Mann M. Mol. Cell. Proteomics, 2012, 11(3): M111.013722
3 Geiger T, Wehner A, Schaab C, Cox J, Mann M. Mol. Cell. Proteomics, 2012, 11(3): M111.014050
4 Gallien S, Duriez E, Crone C, Kellmann M, Moehring T, Domon B. Mol. Cell. Proteomics, 2012, 11(12): 1709-1723
5 Gallien S, Duriez E, Demeure K, Domon B. J. Proteomics, 2013, 81: 148-158
6 Chapman J D, Goodlett D R, Masselon C D. Mass Spectrom. Rev., 2013: 10.1002/mas.21400
7 Law K P, Lim Y P. Expert Rev. Proteomics, 2013, 10(6): 551-566
8 Gillet L C, Navarro P, Tate S, Rost H, Selevsek N, Reiter L, Bonner R, Aebersold R. Mol. Cell. Proteomics, 2012, 11(6): O111.016717
9 Egertson J D, Kuehn A, Merrihew G E, Bateman N W, MacLean B X, Ting Y S, Canterbury J D, Marsh D M, Kellmann M, Zabrouskov V, Wu C C, MacCoss M J. Nat. Methods, 2013, 10(8): 744-746
10 Senko M W, Remes P M, Canterbury J D, Mathur R, Song Q, Eliuk S M, Mullen C, Earley L, Hardman M, Blethrow J D, Bui H, Specht A, Lange O, Denisov E, Makarov A, Horning S, Zabrouskov V. Anal. Chem., 2013, 85(24): 11710-11714
Quantification Analysis of Targeted Proteins in Complex
Sample by Novel Data Independent Acquisition
ZHANG Wei1, Reiko Kiyonami2, JIANG Zheng*1, CHEN Wei1
1(Thermo Fisher Scientific, Shanghai 201206, China)
2(Thermo Fisher Scientific, San Jose, CA, USA)
Abstract Data independent acquisition (DIA) is a novel MS scan mode for quantitative proteomics, acquiring all precursors as well as fragments without any loss of low abundant ions, and breaks the throughput limitation of product ion quantification by highresolution MS. Here we developed three DIA methods on quadrupolelinear ion trapOrbitrap (QqITOT) Tribrid MS, classic DIA, as well as novel wide isolation window SIM scan (WiSIM)DIA and full scanDIA (Full MSDIA). Quantitative analysis of 10 low abundant peptides spiked in Hela cell digest was performed by the three methods for linearity, reproducibility and sensitivity evaluation. The results showed that the LOQs reached amol level (14-435 amol) with good linearity and effective MS/MS confirmation. WiSIMDIA utilizes ultrahigh resolution SIM scan for quantification complementary with classic DIA. The isolation window of Full MSDIA was down to 3 amu, and the data could be directly used for database searching, thus realizing the integration of data dependent acquisition (DDA) and DIA, and avoiding the limitation of using spectra library.
Keywords Orbitrap; Data independent acquisition; Proteomics; Absolute quantification
(Received 24 April 2014; accepted 4 July 2014)
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