DirectMS1: MS/MS-Free Identification of 1000 Proteins of Cellular Proteomes in 5 Minutes

Proteome characterization relies heavily on tandem mass spectrometry (MS/MS) and is thus associated with instrumentation complexity, lengthy analysis time, and limited duty cycle. It was always tempting to implement approaches that do not require MS/MS, yet they were constantly failing to achieve a meaningful depth of quantitative proteome coverage within short experimental times, which is particularly important for clinical or biomarker-discovery applications. Here, we report on the first successful attempt to develop a truly MS/MS-free method, DirectMS1, for bottom-up proteomics. The method is compared with the standard MS/MS-based data-dependent acquisition approach for proteome-wide analysis using 5 min LC gradients. Specifically, we demonstrate identification of 1 000 protein groups for a standard HeLa cell line digest. The amount of loaded sample was varied in a range from 1 to 500 ng, and the method demonstrated 10-fold higher sensitivity. Combined with the recently introduced Diffacto approach for relative protein quantification, DirectMS1 outperforms most popular MS/MS-based label-free quantitation approaches because of significantly higher protein sequence coverage.蛋白质组表征高度依赖串联质谱（MS/MS），因此存在仪器复杂度高、分析耗时冗长、占空比受限等缺陷。长期以来，科研人员始终期望开发无需MS/MS的分析策略，但此类方法始终无法在较短实验周期内实现具备实际意义的深度定量蛋白质组覆盖度，而这一点在临床或生物标志物发现应用中尤为关键。本研究首次成功开发了适用于自下而上蛋白质组学（bottom-up proteomics）的真正无MS/MS分析方法DirectMS1。我们将该方法与基于标准MS/MS的数据依赖性采集策略进行对比，采用5分钟液相色谱（LC）梯度洗脱开展全蛋白质组分析。具体而言，针对标准海拉细胞系酶解样品，本方法可实现1000个蛋白质群组的鉴定。在上样样品量介于1至500纳克（ng）的范围内，该方法的灵敏度提升了10倍。结合近期提出的Diffacto相对蛋白质定量方法，DirectMS1凭借显著更高的蛋白质序列覆盖度，性能优于当前主流的基于MS/MS的无标记定量（label-free quantitation）方法。