Disulfide linkage is critical to protein folding and structural stability. The location of disulfide linkages for antibodies is routinely discovered by comparing the chromatograms of the reduced and non-reduced peptide mapping with location identification confirmed by collision-induced dissociation (CID) mass spectrometry (MS)/MS. However, CID product spectra of disulfide-linked peptides can be difficult to interpret, and provide limited information on the backbone region within the disulfide loop. Here, we applied an electron-transfer dissociation (ETD)/CID combined fragmentation method that identifies the disulfide linkage without intensive LC comparison, and yet maps the disulfide location accurately. The native protein samples were digested using trypsin for proteolysis. The method uses RapiGest SF Surfactant and obviates the need for reduction/alkylation and extensive sample manipulation. An aliquot of the digest was loaded onto a C₄ analytical column. Peptides were gradient-eluted and analyzed using a Thermo Scientific LTQ Orbitrap Elite mass spectrometer for the ETD-triggered CID MS³ experiment. Survey MS scans were followed by data-dependent scans consisting of ETD MS² scans on the most intense ion in the survey scan, followed by 5 MS³ CID scans on the 5 most intense ions in the ETD MS² scan. We were able to identify the disulfide-mediated structural variants A and A/B forms and their corresponding disulfide linkages in an immunoglobulin G2 monoclonal antibody with λ light chain (IgG2λ), where the location of cysteine linkages were unambiguously determined.

二硫键对蛋白质折叠和结构稳定性至关重要。通常通过将还原和未还原肽图谱的色谱图与碰撞诱导解离（CID）质谱（MS）/ MS确认的位置标识进行比较，来常规发现抗体的二硫键位置。但是，二硫键连接的肽的CID产物谱可能难以解释，并且在二硫键环内的骨架区域上提供的信息有限。在这里，我们应用了电子转移解离（ETD）/ CID结合碎片化方法，无需进行密集的LC比较即可识别二硫键，并准确定位二硫键的位置。使用胰蛋白酶消化天然蛋白样品以进行蛋白水解。该方法使用RapiGest SF表面活性剂，无需还原/烷基化和大量样品处理。将消化液的等分试样装到C₄分析柱。使用Thermo Scientific LTQ Orbitrap Elite质谱仪对肽进行梯度洗脱并进行ETD触发的CID MS ³实验分析。调查MS扫描之后进行由ETD MS的数据依赖扫描^2次上在调查扫描的最强离子扫描，接着用5个MS ^3个上在ETD MS 5个最强离子CID扫描²扫描。我们能够在带有λ轻链（IgG2λ）的免疫球蛋白G2单克隆抗体中确定二硫键介导的结构变体A和A / B形式及其对应的二硫键，其中半胱氨酸键的位置是明确确定的。