Remoroza Concepcion A, Burke Meghan C, Yang Xiaoyu, Sheetlin Sergey, Mirokhin Yuri, Markey Sanford P, Tchekhovskoi Dmitrii V, Stein Stephen E
Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, United States.
J Proteome Res. 2022 Oct 7;21(10):2421-2434. doi: 10.1021/acs.jproteome.2c00286. Epub 2022 Sep 16.
We present a mass spectral library-based method for analyzing site-specific N-linked protein glycosylation. Its operation and utility are illustrated by applying it to both newly measured and available proteomics data of human milk glycoproteins. It generates two varieties of mass spectral libraries. One contains glycopeptide abundance distribution spectra (GADS). The other contains tandem mass spectra of the underlying glycopeptides. Both originate from identified glycopeptides in proteolytic digests of human milk and purified glycoproteins, which include tenascin, lactoferrin, and several antibodies. Analysis was also applied to digests of a NIST human milk standard reference material (SRM), leading to a GADS library of N-glycopeptides, enabling the direct comparison of glycopeptide distributions for individual proteins. Tandem spectra underlying each glycopeptide GADS peak are combined to create a second type of library that contains spectra of the underlying glycopeptide spectra. These were acquired by higher-energy (stepped) collision dissociation fragmentation followed by ion-trap fragmentation. Spectra are annotated using MS_Piano, recently reported annotation software. This data, with extensions of a widely used spectral library search and display software, provides accessible mass spectral libraries.
我们提出了一种基于质谱库的方法来分析位点特异性N-连接蛋白糖基化。通过将其应用于新测量的和现有的母乳糖蛋白蛋白质组学数据，说明了该方法的操作和实用性。它生成了两种质谱库。一种包含糖肽丰度分布图（GADS）。另一种包含基础糖肽的串联质谱。两者均源自母乳和纯化糖蛋白（包括腱生蛋白、乳铁蛋白和几种抗体）的蛋白水解消化物中鉴定出的糖肽。分析还应用于美国国家标准与技术研究院（NIST）母乳标准参考物质（SRM）的消化物，从而得到一个N-糖肽的GADS库，能够直接比较单个蛋白质的糖肽分布。每个糖肽GADS峰下的串联质谱被组合起来，创建了另一种类型的库，其中包含基础糖肽谱的质谱。这些质谱是通过高能（阶梯式）碰撞解离碎片化，然后进行离子阱碎片化获得的。使用最近报道的注释软件MS_Piano对质谱进行注释。这些数据，连同广泛使用的光谱库搜索和显示软件的扩展，提供了可访问的质谱库。
