WO2020191846A1 - 脉冲式数据非依赖性采集质谱检测蛋白质组的方法 - Google Patents

脉冲式数据非依赖性采集质谱检测蛋白质组的方法 Download PDF

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WO2020191846A1
WO2020191846A1 PCT/CN2019/084005 CN2019084005W WO2020191846A1 WO 2020191846 A1 WO2020191846 A1 WO 2020191846A1 CN 2019084005 W CN2019084005 W CN 2019084005W WO 2020191846 A1 WO2020191846 A1 WO 2020191846A1
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mass
windows
acquisition
data
mass spectrometry
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郭天南
朱怡
蔡雪
葛伟刚
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西湖大学
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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  • the invention belongs to the technical field of biological sample detection, and specifically relates to a method for detecting proteome by pulsed data-independent acquisition and mass spectrometry.
  • Quantitative proteomics research can explain the causes and laws of the occurrence and development of certain biological phenomena from the protein level. It is useful for life sciences (research objects include humans, animals, plants, microorganisms, and any biological samples containing proteins) and human diseases (including tumors). , Diabetes, etc.) of great significance.
  • life sciences search objects include humans, animals, plants, microorganisms, and any biological samples containing proteins
  • human diseases including tumors. , Diabetes, etc.
  • the differential expression proteomics study of tumor tissue and non-tumor tissue may find that a certain tumor-specific protein is used as a disease marker, which can be used for the early diagnosis, classification and prognosis of tumors.
  • DDA data-dependent acquisition technology
  • DIA data-independent acquisition technology
  • DIA can fragment all detected peptide fragments within the selected retention time (RT) and mass-to-charge ratio (m/z) range, and use the secondary fragment ion information to accurately Quantitative. Since the data generated by DIA is a simple and permanently preserved electronic file, and has high enough repeatability to be repeatedly analyzed when verification is needed in the future, DIA has received more and more attention and application in the research of proteomics. .
  • the key factor of DIA data collection is the number of m/z variable windows collected.
  • the technical problem in the prior art is that under certain liquid phase conditions, due to the limitation of the hardware of the mass spectrometer itself, the number of windows in a mass spectrometry acquisition method is also limited. The limitation of the number of windows leads to precursor ions formed by peptides. In the mass spectrometer, the resolution is not enough and the mutual influence is large, resulting in low resolution of the collected mass spectrometry data and a small number of identified proteins.
  • the present invention provides a method for pulsed data-independent acquisition of mass spectrometry to detect the proteome.
  • the present invention solves the problem that the precursor ions formed by polypeptides are in the mass spectrometer due to the limitation of the number of windows. Due to the insufficient resolution, the mutual influence is large, resulting in the low resolution of the collected data for peptides and the technical problems of fewer types of proteins identified.
  • the method of pulsed data-independent acquisition and mass spectrometry for proteome detection includes the following steps:
  • step (1) Each large window in step (1) is evenly divided into N small windows, and the interval width of the mass-to-charge ratio of the small windows is 2-75 m/z;
  • step (3) Combine M*N acquisition data in the N mass spectrometry scanning methods in step (3), and analyze the acquired data pair.
  • the M in step (1) is an integer greater than 2
  • the range of the overall scanning mass-to-charge ratio of M large windows is 400-1200 m/z
  • the N in step (2) is greater than An integer of 1.
  • the M in step (1) is 24, the overall scanning mass-to-charge ratio of the 24 large windows is in the range of 400-1200 m/z, and the mass-to-charge ratio of the first 20 large windows is in the range The width is 20m/z, and the interval widths of the mass-to-charge ratios of the last four large windows are 60m/z, 80m/z, 120m/z and 140m/z.
  • the N in step (2) is 4, the interval width of the mass-to-charge ratios of the first 80 small windows is 5m/z, and the interval widths of the mass-to-charge ratios of the last 16 small windows are sequentially 4 15m/z, 4 20m/z, 4 30m/z and 4 35m/z.
  • the N in step (2) is 4, the interval width of the mass-to-charge ratios of the first 80 small windows is 10m/z, and the interval widths of the mass-to-charge ratios of the last 16 small windows are sequentially 4 at 30m/z, 4 at 40m/z, 4 at 60m/z and 4 at 70m/z.
  • the present invention has the following beneficial effects:
  • the method for detecting proteome by pulsed data-independent acquisition mass spectrometry provided by the present invention can reduce the mass-to-charge ratio of the divided windows under the same sample preparation conditions and the same number of acquisition data windows.
  • Precursor ions formed by peptides influence each other, increasing the number of injections can obtain higher quality mass spectrometry data, and the number of detected proteins can be significantly doubled.
  • Figure 1 is a schematic diagram of the principle of a data-independent collection method in the prior art
  • Embodiment 1 of the present invention is a schematic diagram of the principle of Embodiment 1 of the present invention.
  • Fig. 3 is a schematic diagram of the principle of Embodiment 2 of the present invention.
  • the method of pulsed data-independent acquisition and mass spectrometry for proteome detection includes the following steps:
  • step (1) The large window in step (1) is evenly divided into 4 small windows to form 96 small windows, the interval width of the mass-to-charge ratio of the first 80 small windows is 5m/z, and the last 16 small windows The interval width of the mass-to-charge ratio is 4 15m/z, 4 20m/z, 4 30m/z and 4 35m/z;
  • step (2) The 96 small windows in step (2) are evenly distributed to the 4 mass spectrometry acquisition methods for scanning; for each method acquisition, all ions in the 24 small windows are selected and fragmented, and the window is detected All the fragments generated by the internal ion, obtain the collected data;
  • step (3) Combine data collected in 96 small windows of the 4 mass spectrometry scanning methods in step (3), and analyze the acquired pairs of collected data.
  • each mass spectrum acquisition method also has 24 acquisition windows.
  • the method of pulsed data-independent acquisition and mass spectrometry for proteome detection includes the following steps:
  • step (1) The large window in step (1) is evenly divided into 4 small windows to form 96 small windows, the interval width of the mass-to-charge ratio of the first 80 small windows is 10m/z, and the last 16 small windows The interval width of the mass-to-charge ratio is 4 30m/z, 4 40m/z, 4 60m/z and 4 70m/z;
  • step (2) The 96 small windows in step (2) are evenly distributed to the 4 mass spectrometry acquisition methods for scanning; for each method acquisition, all ions in the 24 small windows are selected and fragmented, and the window is detected All the fragments generated by the internal ion, obtain the collected data;
  • step (3) Combine data collected in 96 small windows of the 4 mass spectrometry scanning methods in step (3), and analyze the acquired pairs of collected data.
  • This embodiment is built on the basis of embodiment 1.
  • the number of small windows is kept the same, and the width of each small window is doubled, that is, two adjacent windows have an overlap of half the width of the window, and the first 80 windows
  • the width of the window is 10m/z, and there is an overlap of 5m/z between each window.
  • the next 16 windows are 4 windows with a width of 30m/z, and each window has an overlap of 15m/z, 4 A window with a width of 40m/z, each window has an overlap of 20m/z, 4 windows with a width of 60m/z, each window has an overlap of 30m/z, and 3 windows with a width of 70m/z The window and a window with a width of 35m/z have an overlap of 35m/z between each window.
  • the 96 windows are equally distributed to 4 mass spectrometry acquisition methods, each of which is also 24 windows.
  • a 400-405m/z window needs to be added to the fourth mass spectrum acquisition method.
  • the sample uses the method provided in this embodiment to repeatedly collect data 4 times, and finally combines and analyzes the mass spectrum data collected 4 times. It can be considered that the mass spectrum has repeatedly collected data in the range of 400-1200 m/z twice, which is equivalent to complete One technique repeats the experiment.
  • the method for detecting proteome by pulsed mass spectrometry provided by the present invention can also be applied to the variable window DIA acquisition method. It only needs to divide each optimized variable window into several parts and distribute them evenly to different mass spectra. Method, if the sample is repeatedly tested using these mass spectrometry methods, more proteins can be detected.
  • the more proteins were identified.
  • the method for detecting proteome by pulse data independent collection and mass spectrometry provided by the present invention can realize that under the same sample preparation conditions and the same collection data window, by further dividing the mass-to-charge ratio of the window, the peptide precursor ions entering the window can be reduced The mutual influence between them can improve the resolution of the collected data and increase the types of protein identification.

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Abstract

一种脉冲式数据非依赖性采集质谱检测蛋白质组的方法，包括如下步骤：（1）设置M个大窗口，大窗口的质荷比的区间宽度为2-150m/z；（2）将大窗口均匀分隔为N个小窗口，小窗口的质荷比的区间宽度为2-75m/z；（3）将M*N个小窗口均匀分配到N个质谱采集方法中进行扫描，对每一个采集方法，将M个小窗口内所有的离子选择、碎裂，并检测该窗口内离子产生的所有碎片，获得采集数据；（4）合并步骤（3）中N个质谱扫描方法中的M*N次采集数据，并对获得的采集数据对进行分析。该方法减少由多肽形成的前体离子相互影响，从而提高采集的数据分辨率和增加蛋白质鉴定的数量。

Description

脉冲式数据非依赖性采集质谱检测蛋白质组的方法 技术领域
本发明属于生物样品检测技术领域，具体涉及脉冲式数据非依赖性采集质谱检测蛋白质组的方法。
背景技术
定量蛋白质组学研究可从蛋白质层面阐释某种生物现象的发生发展原因与规律，对生命科学(研究对象包括人体、动物、植物、微生物、以及任何含有蛋白质的生物样品)以及人类疾病(包括肿瘤、糖尿病等)的诊疗有重大意义。如对肿瘤组织和非肿瘤组织的差异表达蛋白质组研究，可能发现某种肿瘤特异表达的蛋白质作为疾病的标志物，可用于肿瘤的早期诊断、分型与预后。
在蛋白质组学研究中，人体组织等复杂样本往往有成千上万的蛋白，而目前的质谱技术还不能将所有蛋白同时检测到。目前，蛋白质组最主要的分析方法是数据依赖性采集技术(Data dependent acquisition，DDA)和数据非依赖性采集技术(Data independent acquisition，DIA)。目前，串联质谱非目标化合物分析的主要手段是DDA，该方法易造成低丰度标志物信息的丢失，且存在重复性不佳、定量准确性有待提高的缺陷。近年来，随着质谱硬件技术的快速发展，特别是分辨率和扫描速度的显著提升，DIA应运而生。相较于DDA采集技术，DIA可以对其选择的保留时间(RT)和质荷比(m/z)范围内所有检测到的多肽片段进行碎裂，并通过二级碎片离子信息对其进行精确定量。由于DIA产生的数据是简单且能永久保存的电子文件，并且具备足够高的重复性，可在将来需要验证时反复分析，故而DIA在蛋白质组学的研究中受到越来越广泛的重视与应用。而DIA数据采集的关键因素在于其采集的m/z可变窗口数。现有技术存 在的技术问题是：在一定的液相条件下，由于质谱仪器本身硬件的限制，在一个质谱采集方法中窗口数也有一定的限制，窗口数量的限制导致由多肽形成的前体离子在质谱仪中分离度不够而相互影较大，造成采集的质谱数据分辨率偏低，鉴定的蛋白质数量少。
发明内容
针对上述现有技术中所存在的不足，本发明提供了脉冲式数据非依赖性采集质谱检测蛋白质组的方法，本发明解决了因为窗口数量的限制导致由多肽形成的前体离子在质谱仪中因分离度不够而相互影较大，造成采集的数据对多肽的分辨率偏低，鉴定的蛋白质种类较少的技术问题。
本发明提供的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，具体技术方案如下：
脉冲式数据非依赖性采集质谱检测蛋白质组的方法，包括如下步骤：
(1)设置M个大窗口，所述大窗口的质荷比的区间宽度为2-150m/z；
(2)将步骤(1)中的每一个大窗口均匀分隔为N个小窗口，所述小窗口的质荷比的区间宽度为2-75m/z；
(3)将M*N个小窗口均匀分配到N个质谱采集方法中进行扫描，对每一个采集方法，将M个小窗口内所有的离子选择、碎裂，并检测每个大窗口内离子产生的所有碎片，获得采集数据；
(4)合并步骤(3)中N个质谱扫描方法中的M*N次采集数据，并对所述获得的采集数据对进行分析。
在某些实施方式中，步骤(1)中所述M为大于2的整数，M个大窗口的总体扫描质荷比的区间为400-1200m/z，步骤(2)中所述N为大于1的整数。
在某些实施方式中，步骤(1)中所述M为24，24个大窗口的总体扫描质 荷比的区间为400-1200m/z，所述前20个大窗口的质荷比的区间宽度为20m/z，后四个大窗口的质荷比的区间宽度依次为60m/z，80m/z，120m/z和140m/z。
在某些实施方式中，步骤(2)中所述N为4，所述前80个小窗口的质荷比的区间宽度为5m/z，后16个小窗口质荷比的区间宽度依次为4个15m/z，4个20m/z，4个30m/z和4个35m/z。
在某些实施方式中，步骤(2)中所述N为4，所述前80个小窗口的质荷比的区间宽度为10m/z，后16个小窗口质荷比的区间宽度依次为4个30m/z，4个40m/z，4个60m/z和4个70m/z。
本发明具有以下有益效果：本发明提供的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，可以在相同的样本制备条件，相同的采集数据窗口数下，通过分割窗口的质荷比，减少由多肽形成的前体离子相互影响，增加进样次数，可以得到更高质量的质谱数据，检测到的蛋白数量可以明显增加一倍。
附图说明
图1是现有技术的数据非依赖性采集方法原理示意图；
图2是本发明实施例1的原理示意图；
图3是本发明实施例2的原理示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图1-3，对本发明进一步详细说明。
实施例1
本实施例提供的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，具体技术方案如下：
脉冲式数据非依赖性采集质谱检测蛋白质组的方法，包括如下步骤：
(1)设置24个大窗口，前20个大窗口的质荷比的区间宽度为20m/z，后4个大窗口的质荷比的区间宽度依次为60m/z，80m/z，120m/z和140m/z；
(2)将步骤(1)中的大窗口均匀分隔为4个小窗口，形成96个小窗口，所述前80个小窗口的质荷比的区间宽度为5m/z，后16个小窗口质荷比的区间宽度依次为4个15m/z，4个20m/z，4个30m/z和4个35m/z；
(3)将步骤(2)中的96个小窗口均匀分配到4个质谱采集方法中进行扫描；对每一次方法采集，将24个小窗口内所有的离子选择、碎裂，并检测该窗口内离子产生的所有碎片，获得采集数据；
(4)合并步骤(3)中4个质谱扫描方法中的96个小窗口采集数据，并对所述获得的采集数据对进行分析。
在本实施例中，每个质谱采集方法同样是24个采集窗口。通过合并4次分析共计96个质量窗口采集的所有质谱数据，鉴定到的蛋白数量可比现有技术中的DIA方法增加一倍以上。
实施例2
本实施例提供的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，具体技术方案如下：
脉冲式数据非依赖性采集质谱检测蛋白质组的方法，包括如下步骤：
(1)设置24个大窗口，前20个大窗口的质荷比的区间宽度为20m/z，后4个大窗口的质荷比的区间宽度依次为60m/z，80m/z，120m/z和140m/z；
(2)将步骤(1)中的大窗口均匀分隔为4个小窗口，形成96个小窗口，所述前80个小窗口的质荷比的区间宽度为10m/z，后16个小窗口质荷比的区间宽度依次为4个30m/z，4个40m/z，4个60m/z和4个70m/z；
(3)将步骤(2)中的96个小窗口均匀分配到4个质谱采集方法中进行扫 描；对每一次方法采集，将24个小窗口内所有的离子选择、碎裂，并检测该窗口内离子产生的所有碎片，获得采集数据；
(4)合并步骤(3)中4个质谱扫描方法中的96个小窗口采集数据，并对所述获得的采集数据对进行分析。
本实施例是建立在实施例1的基础上，小窗口的数量保持一致，将每个小窗口的宽度扩展一倍，即相邻的两个窗口均有一半窗口宽度的重叠，前80个窗口的宽度为10m/z，每个窗口之间有5m/z的重叠，接下来16个窗口分别为4个宽度为30m/z的窗口，每个窗口之间有15m/z的重叠，4个宽度为40m/z的窗口，每个窗口之间有20m/z的重叠，4个宽度为60m/z的窗口，每个窗口之间有30m/z的重叠，3个宽度为70m/z的窗口和1个宽度为35m/z的窗口，每个窗口之间有35m/z的重叠。最后将这96个窗口平均分配至4个质谱采集方法，每个质谱采集方法同样是24个窗口。此外，为保证400-405m/z的数据亦被重复扫描一次，需在第4个质谱采集方法上增加一个400-405m/z的窗口。样本运用本实施例提供的方法重复采集了4次数据，最后合并分析该4次采集的质谱数据，即可认为质谱对400-1200m/z范围内的数据重复采集了两次，相当于完成了一次技术重复实验。
此外，本发明提供的脉冲式质谱检测蛋白质组的方法同样可以运用于可变窗口的DIA采集方法中，只需将优化出的每一个可变窗口均分成几份，并平均分配到不同的质谱方法，样本运用这几个质谱方法重复检测，则可以检测到更多的蛋白。
综上所述，在一定的液相条件下，窗口数越多，窗口越小，进入每一个窗口的多肽前体离子数目越少、相互影响就越小，采集的数据分辨率则越高，鉴定到的蛋白质也越多。本发明提供的脉冲数据非依赖性采集质谱检测蛋白质组 的方法，可以实现在相同的样本制备条件、相同的采集数据窗口下，通过进一步分割窗口的质荷比，减少进入窗口的多肽前体离子之间的相互影响，从而提高采集的数据的分辨率，增加蛋白质鉴定的种类。
上述仅为本发明较佳可行实施例，并非是对本发明的限制；本发明也并不限于上述举例。本技术领域的技术人员，在本发明的实质范围内，所作出的变化、改型、添加或替换，也应属于本发明的保护范围。

Claims (5)

  1. 脉冲式数据非依赖性采集质谱检测蛋白质组的方法，其特征在于，包括如下步骤：
    (1)设置M个大窗口，所述大窗口的质荷比的区间宽度为2-150m/z；
    (2)将步骤(1)中的每一个大窗口均匀分隔为N个小窗口，所述小窗口的质荷比的区间宽度为2-75m/z；
    (3)将M*N个小窗口均匀分配到N个质谱采集方法中进行扫描，对每一个采集方法，将M个小窗口内所有的离子选择、碎裂，并检测每个大窗口内离子产生的所有碎片，获得采集数据；
    (4)合并步骤(3)中N个质谱扫描方法中的M*N次采集数据，并对所述获得的采集数据对进行分析。
  2. 根据权利要求1所述的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，其特征在于，步骤(1)中所述M为大于2的整数，M个大窗口的总体扫描质荷比的区间为400-1200m/z，步骤(2)中所述N为大于1的整数。
  3. 根据权利2所述的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，其特征在于，步骤(1)中所述M为24，所述前20个大窗口的质荷比的区间宽度为20m/z，后4个大窗口的质荷比的区间宽度依次为60m/z，80m/z，120m/z和140m/z。
  4. 根据权利要求3所述的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，其特征在于，步骤(2)中所述N为4，所述前80个小窗口的质荷比的区间宽度为5m/z，后16个小窗口质荷比的区间宽度依次为4个15m/z，4个20m/z，4个30m/z和4个35m/z。
  5. 根据权利要求3所述的脉冲式数据非依赖性采集质谱检测蛋白质组的方法，其特征在于，步骤(2)中所述N为4，所述前80个小窗口的质荷比的区 间宽度为10m/z，后16个小窗口质荷比的区间宽度依次为4个30m/z，4个40m/z，4个60m/z和4个70m/z。
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