CLE家族植物小肽激素的质谱碎裂特征研究

Mass Spectrometric Characteristics for CLE Family of Plant Peptide Hormones

CLE(CLAVATA3/Embryo surrounding region)小肽是植物小肽激素中最大的家族,在调控植物分生组织干细胞的分化和分裂中发挥着关键性作用。本工作采用超高效液相色谱-四极杆飞行时间质谱法(UPLC-QTOF MS)研究5种典型CLE小肽的质谱特征,并总结质谱碎裂行为的规律,即CLE小肽的最强准分子离子峰带有2~3个电荷,碰撞诱导解离(CID)主要产生b型和y型离子;在15~20 V最佳碰撞电压下,最容易从脯/羟脯氨酸的氨基、甘氨酸的氨基和羧基端发生断裂形成高丰度的b_(3)和y_(4)等碎片离子。对合成的CLE类似肽(CLEL)的质谱碎裂行为进行预测和实验验证,结果表明,CLEL的质谱碎裂行为与预期完全相符,表明该碎裂规律对CLE家族的小肽具有较强的适用性,可为CLE小肽激素的定性和定量分析提供重要的数据参考。CLE家族结构具有高度保守性,通过对拟南芥中的CLE十二肽基序的质谱碎裂方式和碎片离子进行预测,可根据这些数据信息构建非数据依赖的CLE小肽筛选和鉴定方法,有助于生物学家探寻新的CLE小肽激素。

CLAVATA3/Embryo surrounding region(CLE) peptides, the largest family of plant peptide hormones, is critical in regulating cell differentiation and division in meristematic tissues in plants. In this study, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF MS) equipped with an electrospray ionization source(ESI) was used to investigate the fragmentation mechanism of five typical CLE peptides. The results showed that the strongest parent ion of CLE peptides was two or three fold protonated ion. Under the optimal collision energy(15-20 V), the collision-induced dissociation(CID) of CLE peptides mainly generate b-type and y-type fragment ions due to the cleavage of polypeptide bonds. Based on the MS/MS spectra of peptides standards, it was concluded that highly abundant product ions such as b;and y;ion were easily formed by the cleavage at the Xxx-Pro(Xxx denotes any other amino acid), Xxx-Hyp, Xxx-Gly or Gly-Xxx amide bond. In order to verify above fragment mechanism, a synthesized CLE-like peptide(CLEL) was tested and the mass spectrometry fragmentation behavior of experiment was completely in line with that of predictions. It could be seen that the fragmentation mechanism mentioned above had strong applicability to CLE family peptides. According to the structure-activity relationship revealed by plant physiologist, the CLE residues R1/H1, P4, P7, P9, G6 and H12/N12 are critical for their function. Combined with the cleavage patterns of CLE peptides and the conserved amino acid residues mentioned above, y;, y;, b;, b;, y;and y;ion are valuable for qualitative analysis. So the mass spectrum fragmentation mode and fragment ions(y_(1), b_(3), y_(7), and y;ion) of the CLE dodecapeptide motif in Arabidopsis were predicted, which would provide the necessary scientific basis for the quantitative and qualitative analysis of CLE peptides. Considering the highly conservative structure of the CLE family structure, a data-independent CLE peptides screening and identification strategy based on these mass spectrometry fragmentation behaviors was put forward. Herein, CLEL was used as an unknown peptide to initially verify the feasibility of above screening strategy. The deduced structure by MassLynx software was almost identical with real peptide sequence of CLEL but minor difference in order of the last four residues, which could be easily corrected based on the conserve structure of CLE peptide family. This work will be very helpful for biologists to search those hidden predicted CLE peptides in plant kingdom. Furthermore, the high-abundance product ions of CLE peptides, b;, y;and b;ion, are valuable for quantitative analysis.