含二硫键的蛋白质/多肽的MALDI-TOF MS源内裂解研究

应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法研究含二硫键的人胰岛素与甘精胰岛素酶解液的源内裂解(ISD)。比较了不同基质种类及不同结晶状态对含二硫键的人胰岛素与甘精胰岛素酶解液的源内裂解的影响。结果表明,含二硫键的蛋白质的ISD发生受激光点照射位置的影响,在不同基质与结晶形态的条件下,含二硫键的蛋白质的ISD碎片信息不同。通过分析比较,含二硫键的蛋白质的ISD较容易控制,并且其基质的种类及结晶状态作用很关键。需要获得大量碎片时,使激光照射在样品和阿魏酸(FA)基质形成的大结晶处;不希望出现碎片时,可使用2,4,6-三羟基苯乙酮(THAP)为基质,或使激光照射在样品和其他基质形成的细小均匀结晶处。

In-Source Decay of Disulfide Bond-Containing Proteins/Peptides by MALDI-TOF MS

The disulfide bond is one of the most common post-translational modifications in proteins, of which determination is essential to the comprehensive understanding of protein structures. Disulfide bond analysis has gone through great improvement due to the development of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), especially in terms of speed and sensitivity. In general, the characterization of disulfide-containing peptides is achieved by the reduction of disulfide bonds followed by alkylation. The identification of disulfide/cysteine-containing peptides in digests of proteins is essential to structure elucidation of a protein. In order to present a deep understanding of some phenomena occurring in MALDI MS of disulfide/cysteine-containing proteins, the current work systematically investigated effects of co-crystal size and matrix on MALDI-In Source Decay (ISD) fragmentation of disulfide-containing proteins. Imaging experiments were performed to evaluate the influence of laser shot location on the fragmentation of human insulin and insulin glargine, which were selected as model compounds. The spectrum that was recorded on very finely distributed crystals of FA spot does not exhibit fragments. While a characteristic ‘triplet’ ions with a mass separation of 32 u generated by both symmetric and nonsymmetric cleavages of the disulfide bonds was observed on large crystals. Probably for thermodynamic reasons, the tryptic peptide was subjected to the cleavage of even number of chemical bonds gave rise to radical recombination without reductive ISD. Among several matrices tested including ferulic acid (FA), α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), 3-aminoquinolin (AQ), and 2,4,6-trihydroxy acetophenone (THAP), FA was shown to be a versatile matrix allowing one to induce or prevent ISD according to the location of laser shots. CHCA and SA were found to promote ISD of disulfide-containing proteins, in a location dependent manner. However, unlike in CHCA (or SA), ISD was not systematically observed on all crystals for FA, suggesting differences between the crystallization processes of CHCA (or SA) and FA. Minor fragments were observed when using DHB and AQ as matrices. As for THAP, no fragmentation was observed probably because its three OH-groups in meta-position relative to each other resulted in the nonoccurrence of redox reaction. The studies provide insights into the experimental conditions required for determination of disulfide-containing protein by MALDI MS and are helpful for mass spectrum interpretation, opening the way to more rational studies of disulfide/cysteine-containing proteins by MALDI mass spectrometry.