New Algorithm for ¹⁵N/¹⁴N Quantitation with LC−ESI−MS Using an LTQ-FT Mass Spectrometer

A new algorithm (QN) for the 15N /14N quantitation of relative protein abundances in complex proteomic samples is described. QN takes advantage of the high resolution, mass accuracy and throughput of the hybrid mass spectrometer LTQ-FT MS. Peptide quantitation is based on MS peak intensity (measured in the FT MS), while peptide identification is performed in the MS/MS mode (measured in the LTQ linear ion trap). Accuracy of the protein abundance is enhanced by a novel scoring procedure, allowing filtering of less reliable measurements of peptide abundances. The performance of QN is illustrated in the relative quantitative analysis of M. acetivorans C2A cultures grown with carbon monoxide vs methanol as substrate. Roughly 1000 proteins were quantitated with an average CV of 9% for the protein abundance ratios. QN performs quantitation without manual intervention, does not require high processing power, and generates files compatible with the Guidelines for Proteomic Data Publication. Keywords: quantitative proteomics • LC−MS • metabolic labeling • software • M. acetivorans本研究报道了一种用于复杂蛋白质组样品中相对蛋白质丰度15N/14N定量分析的新型算法（QN）。该算法充分利用混合型质谱仪LTQ-FT MS的高分辨率、高质量精度与高通量分析能力。肽段定量基于傅里叶变换质谱（FT MS）所测得的质谱峰强度，而肽段鉴定则通过LTQ线性离子阱的串联质谱（MS/MS）模式完成。通过全新的评分流程可对可信度较低的肽段丰度测量结果进行过滤，从而提升蛋白质丰度定量的准确性。 本研究以一氧化碳与甲醇为底物分别培养的马氏甲烷八叠球菌（M. acetivorans）C2A菌株的相对定量分析为例，验证了QN算法的性能。该算法可对约1000种蛋白质进行定量分析，蛋白质丰度比的平均变异系数（CV）为9%。QN可实现全自动定量，无需高性能计算资源，且生成的文件符合《蛋白质组学数据发表指南（Guidelines for Proteomic Data Publication）》的格式要求。 关键词：定量蛋白质组学 • 液相色谱-质谱联用（LC−MS） • 代谢标记 • 软件 • 马氏甲烷八叠球菌（M. acetivorans）