Single-Shot Capillary Zone Electrophoresis–Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample

The
dependence of capillary zone electrophoresis (CZE) separations
on the charge state of the analyte is useful for the analysis of many
post-translational modifications in proteins. In this work, we coupled
CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced
peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated
capillary with very low electroosmotic flow was used for the separation.
The optimal injection volume was between 100 and 150 nL of a solution
of phosphopeptides in 30 mM ammonium bicarbonate (pH 8.2) buffer,
which produces a dynamic pH junction sample injection. Larger injection
volumes resulted in serious peak broadening and decreased numbers
of phosphopeptide identifications. The optimized system identified
4405 phosphopeptides from 220 ng of enriched phosphopeptides from
mouse brain, which represents the state-of-the-art result for single-shot
CZE–ESI–MS/MS-based phosphoproteome analysis. We found
that the migration time for phosphopeptides is much longer than that
for non-phosphopeptides and increased along with the number of phosphorylation
sites on the peptides, as expected for the additional negative charges
associated with the phosphate groups. We also investigated the phosphorylation
site motifs; a number of motifs appeared in the CZE–ESI–MS/MS
data but not in LC–ESI–MS/MS data, which suggested the
complementary performance of the techniques. The data are available
via ProteomeXchange with identifier PXD012888.

毛细管区带电泳（capillary zone electrophoresis, CZE）的分离效果依赖于分析物的电荷状态，该特性可用于蛋白质多种翻译后修饰的分析。本研究将毛细管区带电泳与搭载先进峰判定算法的Orbitrap Fusion Lumos Tribrid质谱平台联用，用于磷酸化蛋白质组学分析。实验采用电渗流极低的线性聚丙烯酰胺涂层毛细管完成分离。最优进样体积为100~150 nL的磷酸肽溶液，该溶液以30 mM碳酸氢铵（pH 8.2）为缓冲液，可实现动态pH交汇进样。进样体积过大会引发严重的峰展宽，并降低磷酸肽的鉴定数量。经优化的分析体系可从220 ng富集得到的小鼠脑组织磷酸肽中，鉴定出4405条磷酸肽，这是当前基于单次毛细管区带电泳-电喷雾电离-串联质谱法的磷酸化蛋白质组分析领域的顶尖成果。研究发现，磷酸肽的迁移时间远长于非磷酸肽，且随肽段上磷酸化位点数量增加而延长，这与磷酸基团携带额外负电荷的预期规律相符。我们还对磷酸化位点基序展开了研究：部分基序仅在毛细管区带电泳-电喷雾电离-串联质谱数据中检出，而未在液相色谱-电喷雾电离-串联质谱数据中出现，这表明两种技术具有互补性。本研究相关数据可通过标识符为PXD012888的蛋白质组交换数据库（ProteomeXchange）获取。