Discovery of Protein Modifications Using Differential Tandem Mass Spectrometry Proteomics

Recent studies have revealed diverse
amino acid, post-translational,
and noncanonical modifications of proteins in diverse organisms and
tissues. However, their unbiased detection and analysis remain hindered
by technical limitations. Here, we present a spectral alignment method
for the identification of protein modifications using high-resolution
mass spectrometry proteomics. Termed SAMPEI for spectral alignment-based
modified peptide identification, this open-source algorithm is designed
for the discovery of functional protein and peptide signaling modifications,
without prior knowledge of their identities. Using synthetic standards
and controlled chemical labeling experiments, we demonstrate its high
specificity and sensitivity for the discovery of substoichiometric
protein modifications in complex cellular extracts. SAMPEI mapping
of mouse macrophage differentiation revealed diverse post-translational
protein modifications, including distinct forms of cysteine itaconatylation.
SAMPEI’s robust parametrization and versatility are expected
to facilitate the discovery of biological modifications of diverse
macromolecules. SAMPEI is implemented as a Python package and is available
open-source from BioConda and GitHub (https://github.com/FenyoLab/SAMPEI).

近期已有研究在多种生物及组织中，发现蛋白质存在多样化的氨基酸修饰、翻译后修饰（post-translational）与非经典修饰。然而，受限于当前技术瓶颈，此类修饰的无偏检测与分析仍存在较大阻碍。本研究提出一种基于高分辨质谱蛋白质组学的蛋白质修饰鉴定光谱比对方法。该开源算法被命名为SAMPEI（基于光谱比对的修饰肽段鉴定，Spectral Alignment-based Modified Peptide Identification），旨在无需预先知晓修饰类型的前提下，挖掘具有功能活性的蛋白质与肽段信号修饰。本研究通过合成标准品与受控化学标记实验，证实该算法在复杂细胞提取物中挖掘亚化学计量蛋白质修饰时，具备极高的特异性与灵敏度。通过对小鼠巨噬细胞分化过程的SAMPEI分析，本研究发现了多样化的蛋白质翻译后修饰，包括多种不同形式的半胱氨酸衣康酰化修饰。SAMPEI具备稳健的参数化能力与广泛的适用性，有望助力多种大分子生物修饰的挖掘工作。SAMPEI以Python包的形式实现，可通过BioConda及GitHub（https://github.com/FenyoLab/SAMPEI）开源获取。