Center for Proteomics & Metabolomics, Leiden University Medical Center, Albinusdreef 2, Leiden 2333 ZA, The Netherlands.
Department of Chemistry, Nottingham Trent University, Nottingham NG11 0JN, U.K.
Anal Chem. 2020 Sep 15;92(18):12429-12436. doi: 10.1021/acs.analchem.0c02198. Epub 2020 Sep 1.
The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above / 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase the detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found good coverage of the peptide chain termini starting from c'2 and z'2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the / range 46-13 500 showed an increased sequence coverage for three standard proteins, namely, myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary.
各种离子化和碎片化技术的发展对于将质谱（MS）确立为蛋白质特征分析的强大工具至关重要。其中一个例子是基质辅助激光解吸/电离（MALDI）与源内碎裂（ISD）相结合，无需进行蛋白水解即可对大蛋白质的 N 端和 C 端区域进行作图。正离子模式 ISD 片段通常在 1000 以上的质量区域中被分配，而 MALDI 基质离子通常会阻碍较小的单电荷片段的检测。傅里叶变换离子回旋共振（FT-ICR）MS 提供的超高分辨率部分克服了这一限制，但为了进一步提高对较小片段的检测，我们重新研究了负离子模式 MALDI-ISD 的应用，并发现从 c'2 和 z'2 片段离子开始，肽链末端得到了很好的覆盖。我们首次证明了正负离子 MALDI FT-ICR MS 的组合是一种有用的工具，可以改善 mAb 的表征。两种离子模式的不同特异性允许我们选择性地以更高的灵敏度覆盖 mAb 的轻链和重链序列。对正离子和负离子模式 MALDI-ISD FT-ICR MS 在 46-13500 范围内的综合评估表明，三种标准蛋白质（即肌红蛋白、SiLuLite mAb 和 NIST mAb）的序列覆盖率有所提高。两种离子模式获得的数据在某些方面是互补的。
