Optimizing Linear Ion-Trap Data-Independent Acquisition toward Single-Cell Proteomics.
Optimizing Linear Ion-Trap Data-Independent Acquisition toward Single-Cell Proteomics.
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DOI：
10.1021/acs.analchem.3c00842
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发表时间：
2023-06
影响因子：
7.4
通讯作者：
Teeradon Phlairaharn;Zilu Ye;Elena Krismer;A. Pedersen;M. Pietzner;J. Olsen;E. Schoof;B. Searle
中科院分区：
文献类型：
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作者：
Teeradon Phlairaharn;Zilu Ye;Elena Krismer;A. Pedersen;M. Pietzner;J. Olsen;E. Schoof;B. Searle
线性离子阱（LIT）是一种经济实惠的、稳健的质谱仪，其提供快速扫描速度和高灵敏度，其中其主要缺点是与更常用的飞行时间或轨道阱（OT）质量分析仪相比质量精度较差。先前利用LIT进行低输入蛋白质组学分析的努力仍然依赖于用于收集前体数据的内置OT或基于OT的文库生成。在这里，我们展示了低输入蛋白质组学的LIT作为所有质谱（MS）测量（包括文库生成）的独立质量分析仪的潜在多功能性。为了测试这种方法，我们首先优化了LIT数据采集方法，并在有和没有包埋肽的情况下进行了无文库搜索，以评估检测和定量准确度。然后，我们生成了基质匹配的校准曲线，以仅使用10 ng起始材料估计定量下限。虽然LIT-MS 1测量提供的定量准确度较差，但LIT-MS 2测量在色谱柱上的定量准确度低至0.5 ng。最后，我们优化了从低输入材料生成光谱库的合适策略，我们使用该策略通过LIT-DIA使用从少至40个细胞生成的基于LIT的库来分析单细胞样品。
A linear ion trap (LIT) is an affordable, robust mass spectrometer that provides fast scanning speed and high sensitivity, where its primary disadvantage is inferior mass accuracy compared to more commonly used time-of-flight or orbitrap (OT) mass analyzers. Previous efforts to utilize the LIT for low-input proteomics analysis still rely on either built-in OTs for collecting precursor data or OT-based library generation. Here, we demonstrate the potential versatility of the LIT for low-input proteomics as a stand-alone mass analyzer for all mass spectrometry (MS) measurements, including library generation. To test this approach, we first optimized LIT data acquisition methods and performed library-free searches with and without entrapment peptides to evaluate both the detection and quantification accuracy. We then generated matrix-matched calibration curves to estimate the lower limit of quantification using only 10 ng of starting material. While LIT-MS1 measurements provided poor quantitative accuracy, LIT-MS2 measurements were quantitatively accurate down to 0.5 ng on the column. Finally, we optimized a suitable strategy for spectral library generation from low-input material, which we used to analyze single-cell samples by LIT-DIA using LIT-based libraries generated from as few as 40 cells.