Extending Proteome Coverage by Combining MS/MS Methods and a Modified Bioinformatics Platform Adapted for Database Searching of Positive and Negative Polarity 193 nm Ultraviolet Photodissociation Mass Spectra

To
extend proteome coverage obtained from bottom-up mass spectrometry
approaches, three complementary ion activation methods, higher energy
collision dissociation (HCD), ultraviolet photodissociation (UVPD),
and negative mode UVPD (NUVPD), are used to interrogate the tryptic
peptides in a human hepatocyte lysate using a high performance Orbitrap
mass spectrometer. The utility of combining results from multiple
activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth
and breadth of proteome coverage. This study also benchmarks a new
version of the Byonic algorithm, which has been customized for database
searches of UVPD and NUVPD data. Searches utilizing the customized
algorithm resulted in over 50% more peptide identifications for UVPD
and NUVPD tryptic peptide data sets compared to other search algorithms.
Inclusion of UVPD and NUVPD spectra resulted in over 600 additional
protein identifications relative to HCD alone.

为拓展自下而上质谱法（bottom-up mass spectrometry）所能实现的蛋白质组覆盖范围，本研究采用三种互补的离子活化方法——高能碰撞解离（higher energy collision dissociation, HCD）、紫外光解离（ultraviolet photodissociation, UVPD）以及负模式紫外光解离（negative mode UVPD, NUVPD），借助高性能Orbitrap质谱仪对人肝细胞裂解液中的胰蛋白酶肽进行分析。本研究针对多活化技术联用（HCD+UVPD+NUVPD）的结果效用，从蛋白质组覆盖的总深度与广度两个维度展开分析。此外，本研究还对一款针对紫外光解离与负模式紫外光解离数据的数据库检索任务定制的新版Byonic算法（Byonic algorithm）开展了基准性能测试。相较于其他检索算法，使用该定制化算法开展的检索可使UVPD与NUVPD胰蛋白酶肽数据集的肽段鉴定数量提升50%以上。相较于仅使用HCD的实验方案，纳入UVPD与NUVPD光谱后可额外实现600余种蛋白质的鉴定。