Advancing Intact Protein Quantitation with Updated Deconvolution Routines.
Advancing Intact Protein Quantitation with Updated Deconvolution Routines.
复制标题
通过更新的解卷积程序推进完整蛋白质定量。
DOI：
10.1021/acs.analchem.3c02345
复制
发表时间：
2023
影响因子：
7.4
通讯作者：
Durbin,KennethR
中科院分区：
文献类型：
--
作者：
Robey,MatthewT;Utley,Daisha;Greer,JosephB;Fellers,RyanT;Kelleher,NeilL;Durbin,KennethR
通过质谱分析完整蛋白质可以直接定量样品中存在的特定蛋白质形态，并且是生物制药和学术研究中越来越重要的工具。从质谱中解释和定量完整的蛋白质物种通常涉及许多挑战，包括质量反褶积和峰处理，以及确定最佳的光谱平均参数和将质量与理论蛋白质形态相匹配。这些步骤中的每一步都可能带来信息障碍，因为参数通常需要专门针对数据集进行定制。为了减少完整质量反褶积数据分析的负担，我们在广泛使用的“滑动窗口”质量反褶积技术的基础上增加了几个额外的概念。首先，我们发现如何平均光谱和光谱窗口的重叠可以调整到有利于灵敏度或速度。发现多窗口平均方法是增加质量检测的最有效方法，并使检测到的质量数量增加了50倍。我们还开发了一种有针对性的特征查找程序，将灵敏度提高了50倍，将重复间的变异系数降低了50%，并通过检测到的时间点增加了3倍，提高了质量洗脱谱的质量。最后，我们进一步完善了现有的方法，通过光谱拟合来对检测到的具有潜在变形形式的质量进行注释，以进行可能的变形形式家族修饰和网络查看。这些变形形式注释方法最终产生了一种更准确的方法，从完整的质量数据中找到相关的，但以前未知的变形形式。总之，这些量化工作流程的改进提高了从完整蛋白质质谱分析中获得的信息。
Analysis of intact proteins by mass spectrometry enables direct quantitation of the specific proteoforms present in a sample and is an increasingly important tool for biopharmaceutical and academic research. Interpreting and quantifying intact protein species from mass spectra typically involves many challenges including mass deconvolution and peak processing as well as determining optimal spectral averaging parameters and matching masses to theoretical proteoforms. Each of these steps can present informatic hurdles, as parameters often need to be tailored specifically to the data sets. To reduce intact mass deconvolution data analysis burdens, we built upon the widely used “sliding window” mass deconvolution technique with several additional concepts. First, we found that how spectra are averaged and the overlap in spectral windows can be tuned to favor either sensitivity or speed. A multiple window averaging approach was found to be the most effective way to increase mass detection and yielded a >2-fold increase in the number of masses detected. We also developed a targeted feature-finding routine that boosted sensitivity by >2-fold, decreased coefficient of variation across replicates by 50%, and increased the quality of mass elution profiles through 3-fold more detected time points. Lastly, we furthered existing approaches for annotating detected masses with potential proteoforms through spectral fitting for possible proteoform family modifications and network viewing. These proteoform annotation approaches ultimately produced a more accurate way of finding related, but previously unknown proteoforms from intact mass-only data. Together, these quantitation workflow improvements advance the information obtainable from intact protein mass spectrometry analyses.