Optimization and Modeling of Quadrupole Orbitrap Parameters for Sensitive Analysis toward Single-Cell Proteomics

Single-cell proteomics represents a field of extremely sensitive proteomic analysis, owing to the minute amount of yet complex proteins in a single cell. Without amplification potential as of nucleic acids, single-cell mass spectrometry (MS) analysis demands special instrumentation running with optimized parameters to maximize the sensitivity and throughput for comprehensive proteomic discovery. To facilitate such analysis, we here investigated two factors critical to peptide sequencing and protein detection in shotgun proteomics, i.e. precursor ion isolation window (IW) and maximum precursor ion injection time (ITmax), on an ultrahigh-field quadrupole Orbitrap (Q-Exactive HF). Counterintuitive to the frequently used proteomic parameters for bulk samples (>100 ng), our experimental data and subsequent modeling suggested a universally optimal IW of 4.0 Th for sample quantity ranging from 100 ng to 1 ng, and a sample-quantity dependent ITmax of more than 250 ms for 1-ng samples. Compared with the benchmark condition of IW = 2.0 Th and ITmax = 50 ms, our optimization generated up to 300% increase to the detected protein groups for 1-ng samples. The additionally identified proteins allowed deeper penetration of proteome for better revealing crucial cellular functions such as signaling and cell adhesion. We hope this effort can prompt single-cell and trace proteomic analysis and enable a rational selection of MS parameters.

单细胞蛋白质组学（single-cell proteomics）是一类极具灵敏度的蛋白质组分析领域，其核心挑战源于单个细胞内蛋白质含量极微且组成复杂。由于无法像核酸那样实现扩增，单细胞质谱（mass spectrometry, MS）分析需要采用经过参数优化的专用仪器，以最大化分析灵敏度与通量，实现全面的蛋白质组鉴定。为助力此类分析，本研究在超高场四极杆轨道阱（ultrahigh-field quadrupole Orbitrap, Q-Exactive HF）质谱仪上，探究了鸟枪法蛋白质组学中肽段测序与蛋白质鉴定的两个关键因素：前体离子隔离窗口（precursor ion isolation window, IW）与最大前体离子注入时间（maximum precursor ion injection time, ITmax）。相较于常规应用于大量样本（>100 ng）的蛋白质组学参数，本研究的实验数据与后续建模结果表明：对于1 ng至100 ng的样本量，通用最优前体离子隔离窗口为4.0 Th；而1 ng样本的最大前体离子注入时间需大于250 ms，且该参数随样本量变化而调整。相较于基准参数设置（IW=2.0 Th、ITmax=50 ms），本研究的优化方案可使1 ng样本的鉴定蛋白质组群数量增幅最高达300%。新增鉴定出的蛋白质可实现更深度的蛋白质组覆盖，从而更好地揭示信号传导、细胞黏附等关键细胞功能。本研究希望能够推动单细胞与微量蛋白质组分析领域的发展，并为质谱参数的合理选择提供参考依据。