Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8362, Gaithersburg, Maryland 20899, United States.
J Proteome Res. 2021 Mar 5;20(3):1612-1629. doi: 10.1021/acs.jproteome.0c00823. Epub 2021 Feb 8.
This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb, a reference material of the humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced and non-reduced samples under neutral and weakly basic conditions followed by nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS). Spectra of peptides containing disulfide bonds are identified by both MS1 ion and MS2 fragment ion data in order to completely map all the disulfide linkages in the NISTmAb. This led to the detection of 383 distinct disulfide-linked peptide ions, arising from fully tryptic cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low-energy collision dissociation were examined. These include (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from breakages of two peptide bonds or both peptide and disulfide bonds. Automated annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the nine native disulfide bonds were identified along with 86 additional disulfide linkages arising from disulfide bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 disulfide-linked peptide spectra was created from this analysis and is publicly available for free download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of "hard-to-find" disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in proteomics studies. Data are available via ProteomeXchange with identifier PXD023358.
这项工作提出了用于识别和创建源自 NISTmAb（人源化 IgG1k 单克隆抗体 RM 8671 的参考物质）的二硫键连接肽的质谱文库的方法。分析涉及中性和弱碱性条件下部分还原和非还原的样品，然后进行纳流液相色谱串联质谱（LC-MS/MS）分析。通过 MS1 离子和 MS2 碎片离子数据来识别含有二硫键的肽谱，以完全绘制 NISTmAb 中的所有二硫键连接。这导致检测到 383 种独特的二硫键连接肽离子，这些肽来自完全胰蛋白酶切割、缺失切割、不规则切割、复杂的 Met/Trp 氧化混合物和金属加合物。研究了低能碰撞解离下二硫键的断裂特征。这些特征包括：（1）肽键断裂，二硫键保持完整；（2）二硫键断裂，通常导致广泛的碎片化；（3）两条肽键或肽键和二硫键断裂产生的双断裂产物。各种复杂 MS/MS 片段的自动注释使得能够高度置信地鉴定二硫键连接的肽。鉴定了含有九个天然二硫键的肽，以及 86 个来自二硫键重排的额外二硫键连接。通过优化消化条件，几乎完全消除了重排二硫键的存在。从该分析中创建了一个包含 702 个二硫键连接肽谱的精选光谱文库，并免费提供公开下载。由于所有 IgG1 抗体都具有相同的恒定区，因此生成的文库可用于轻松鉴定“难以找到”的二硫键结合肽。此外，我们表明，可以鉴定来自人血清中 IgG1 蛋白的此类肽，从而作为蛋白质组学研究中监测蛋白质还原完全性的一种手段。数据可通过 ProteomeXchange 以标识符 PXD023358 获得。
