Mertz Keaton L, Serrano Lia R, Sinitcyn Pavel, Coon Joshua J
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Morgridge Institute for Research, Madison, Wisconsin 53515, United States.
J Am Soc Mass Spectrom. 2025 Aug 8. doi: 10.1021/jasms.5c00110.
Data-independent acquisition (DIA) mass spectrometry facilitates high-throughput, reproducible bottom-up proteomic analyses. Typically, DIA methods coselect multiple precursor ions within a wide selection window. These precursors are simultaneously fragmented, superimposing the product ion signals into a complex chimeric spectrum. A method for varying the quadrupole selection width over the ion accumulation period is described. This method couples the intensity of a product ion to the mass of its precursor ion. By overlapping consecutive selection windows, scan-to-scan product ion intensity profiles can be used to infer precursor mass. We assess the method's sensitivity to quadrupole width, accumulation time, and mass-to-charge range using internal fluoranthene calibrant and FlexMix calibration solution with Q-Orbitrap configured mass analyzers. Additionally, we explore usability of the described technique on a tryptic-digest monoclonal antibody sample, including both direct infusion and liquid chromatography of the sample. With direct infusion, product ions from two precursors separated by 1 thomson (Th) are resolved with this method using 10 Th windows with 5 Th overlap. The product ions are associated within 0.3 Th of their respective precursor ion's /. Therefore, product ion spectra have a precursor ion / resolving power of ∼33.
数据非依赖型采集（DIA）质谱技术有助于实现高通量、可重复的自下而上蛋白质组学分析。通常，DIA方法在较宽的选择窗口内共同选择多个前体离子。这些前体离子同时裂解，将产物离子信号叠加成一个复杂的嵌合谱图。本文描述了一种在离子积累期间改变四极杆选择宽度的方法。该方法将产物离子的强度与其前体离子的质量相关联。通过重叠连续的选择窗口，逐次扫描的产物离子强度分布可用于推断前体质量。我们使用内标荧蒽和FlexMix校准溶液，在配备Q-轨道阱的质量分析器上评估该方法对四极杆宽度、积累时间和质荷比范围的灵敏度。此外，我们还探讨了该技术在胰蛋白酶消化的单克隆抗体样品上的适用性，包括样品的直接进样和液相色谱分析。直接进样时，使用10 Th的窗口且重叠5 Th，该方法可分辨出相隔1道尔顿（Da）的两个前体离子产生的产物离子。产物离子与其各自前体离子的质荷比相差在0.3 Th以内。因此，产物离子谱图的前体离子质荷比分辨能力约为33。
