Title：Leveraging a higher duty cycle DIA acquisition on a novel QTOF for enhanced proteomics analysis

Journal：Sciex Application note

Date：2021

Abstract:

We used a research version of ZenoTOF 7600 system coupled to Evosep One LC system to evaluate gains in protein identifications at various HeLa peptide loads when using variable window Zeno SWATH relative to SWATH acquisition. EvoSep was operated at throughputs of 200 SPD (samples per day, 5.6 min gradient), 100 SPD (11.5 min gradient), 60 SPD (21 min gradient) and nano-flow 30 SPD (44 min gradient). When we utilize the Zeno trap to increase the duty cycle at the MS/MS level to over 90% in Zeno SWATH mode (Zeno trap turned ON), the quality of the MS/MS spectra increases greatly relative to that acquired in SWATH acquisition

alone. Analyzing the same sample loads with Zeno SWATH rather than SWATH acquisition, we obtain 1.5-2.4x more protein group identifications at 20% CV threshold,1.8-3.2x more at 10% CV threshold and 1.2-1.5x increase in overall identifications at low (25-50 ng) protein loads at all SPD throughputs. At higher protein loads (200-500 ng), the increase in protein group identifications is 1.3-1.5x at 20% CV threshold and 1.4-1.8x at 10% CV threshold. With Zeno SWATH we are able to identify over 8400 protein groups for 200 ng and 500 ng HeLa load for the 30 SPD throughput with 80-90% of identifications at 20% CV, or 7600-7700 proteins at 60 SPD with comparable CVs. The ‘library-free’ search approach for 500 ng load at 30 SPD identified 7700 protein groups, comparable number of IDs at CV thresholds relative to spectra-library approach and demonstrates the ease-of-use of the Zeno SWATH methodology in high-throughput proteomics.

1.样品制备:

冻干的HeLa消化物购自Thermo Fisher Scientific，用95%缓冲液A(含0.1%甲酸的水)和5%缓冲液B(含0.1%甲酸的乙腈)复溶至0.1µg/µL的工作浓度。25 ng上样量对应的浓度 1.25 ng/ µL，50 ng上样量对应的浓度是2.5 ng/ µL，200 ng上样量对应的浓度是10 ng/ µL，500 ng上样量对应的浓度是25 ng/ µL。将不同浓度的20 µL样品加载到Evotips中。

2. 色谱方法:

Evosep One (Evosep，丹麦)以Buffer A和B作为流动相，并以200 SPD(每天样品，5.6分钟梯度)、100 SPD (11.5分钟梯度)、60 SPD (21.0分钟梯度)和30 SPD (44.0分钟梯度)的通量运行。200 SPD方法使用4cm×150µm内径、1.9 µm颗粒柱(EV 1107);100 SPD和60 SPD方法使用8 cm x 150 µm内径、1.5 µm颗粒柱(EV1109)，30 SPD方法使用15 cm x 150 µm内径、1.9 µm的色谱柱(EV1106)

3. 质谱方法:

ZenoTOF 7600系统使用OptiFlow源在SWATH和Zeno SWATH采集模式下运行。200 SPD、100 SPD和60 SPD以微流运行，30 SPD以纳流运行。

4. 数据处理:

使用DIA-NN(1.8版)软件处理Zeno SWATH和SWATH数据，使用内部生成的HeLa和K562细胞系的分级数据库(11269个蛋白质组和169395个前体)或SwissProt-Human canonical+ isoform (2021年1月)FASTA数据库进行无库搜索。pg.matrix.tsv和pr.matrix.tsv用于报告蛋白质组和前体，并用于计算%CV阈值的IDs。

1.蛋白质组

SWATH和Zeno SWATH数据采集在所有(200、100、60、30) SPD方法下的对比(Figure 1-4)。

• 蛋白质含量较低时: 25 ng上样量在CV<20%和CV<10% 时，蛋白质组数量分别增加了2.2-2.4倍(120-140%)和2.4-3.2倍(140-220%); 50 ng上样量在CV<20%和CV<10%时，蛋白质组数量分别增加了1.5-1.9倍(50-90%)和1.8-2.3倍(80-130%);

• 蛋白质含量较高时: 200 ng上样量在CV<20%和CV<10% 时，蛋白质组数量分别增加1.3-1.5倍(30-50%)和1.4-1.8倍(40-80%);500 ng上样量在CV<20%和CV<10% 时，蛋白质组数量分别增加了1.3-1.4倍(30-40%)和1.5-1.7倍(50-70%)。

1.Zeno SWATH能够实现更高数量的蛋白质鉴定和定量(2-3倍)，特别是相对于SWATH采集更低的蛋白质上样量;

2.对于低上样量样品非常重要，例如单细胞、激光捕获显微切割(LCM)、福尔马林固定石蜡包埋(FFPE)工作流程;

3.可变窗口条带采集的速度和选择性以及Zeno条带解决了高通量环境中的需求;

4.Zeno SWATH和无文库方法展示了DIA方法在高通量蛋白质组工作流程中的易用性。

1. Chernushevich I., Loboda A. J Am Soc Mass Spectrom., 20 (7), 2009.

2. Demichev, V., et al. Nature Methods, 17 (1), 2020.