Mass Spectrometric Characteristics for CLE Family of Plant Peptide Hormones

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 b₃ and y₄ 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, y₁, y₄, b₅, b₆, y₆ and y₇ ion are valuable for qualitative analysis. So the mass spectrum fragmentation mode and fragment ions (y₁, b₃, y₄, and y₇ 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, b3, y4 and b6 ion, are valuable for quantitative analysis.