Bioinformatics Program, Boston University, United States.
Dept. of Biochemistry, Boston University, United States.
Curr Opin Struct Biol. 2022 Jun;74:102371. doi: 10.1016/j.sbi.2022.102371. Epub 2022 Apr 19.
Biosynthetic enzymes in the secretory pathway create distributions of glycans at each glycosite that elaborate the biophysical properties and biological functions of glycoproteins. Because the biosynthetic glycosylation reactions do not go to completion, each protein glycosite is heterogeneous with respect to glycosylation. This heterogeneity means that it is not sufficient to measure protein abundance in omics experiments. Rather, it is necessary to sample the distribution of glycosylation at each glycosite to quantify the changes that occur during biological processes. On the one hand, the use of data-dependent acquisition methods to sample glycopeptides is limited by the instrument duty cycle and the missing value problem. On the other, stepped window data-independent acquisition samples all precursors, but ion abundances are limited by duty cycle. Therefore, the ability to quantify accurately the flux in glycoprotein glycosylation that occurs during biological processes requires the exploitation of emerging mass spectrometry technologies capable of deep, comprehensive sampling and selective high confidence assignment of the complex glycopeptide mixtures. This review summarizes recent technical advances and mass spectral glycoproteomics analysis strategies and how these developments impact our ability to quantify the changes in glycosylation that occur during biological processes. We highlight specific improvements to glycopeptide characterization through activated electron dissociation, ion mobility trends and instrumentation, and efficient algorithmic approaches for glycopeptide assignment. We also discuss the emerging need for unified standards to enable interlaboratory collaborations and effective monitoring of structural changes in glycoproteins.
生物合成酶在分泌途径中创造了每个糖基化位点的聚糖分布，这些分布详细阐述了糖蛋白的生物物理性质和生物学功能。由于生物合成糖基化反应没有完成，因此每个蛋白质糖基化位点在糖基化方面都是异质的。这种异质性意味着在组学实验中仅测量蛋白质丰度是不够的。相反，有必要对每个糖基化位点的糖基化分布进行采样，以量化在生物过程中发生的变化。一方面，使用基于数据的采集方法来采样糖肽受到仪器工作周期和缺失值问题的限制。另一方面，分步窗口的数据独立采集方法可以采样所有前体，但离子丰度受到工作周期的限制。因此，要准确地定量生物过程中糖蛋白糖基化发生的通量，需要利用新兴的质谱技术，这些技术能够深入、全面地采样，并选择性地对复杂的糖肽混合物进行高置信度分配。这篇综述总结了最近的技术进展和质谱糖蛋白质组学分析策略，以及这些进展如何影响我们定量生物过程中发生的糖基化变化的能力。我们强调了通过活性电子解离、离子淌度趋势和仪器以及用于糖肽分配的高效算法方法对糖肽特征的具体改进。我们还讨论了新兴的对统一标准的需求，以实现实验室间的合作以及对糖蛋白结构变化的有效监测。
