One of significant characteristics of matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry ( MALD-TOF/TOF ) high-energy collision induced dissociation ( CID ) is to produce abundant immonium ( IM ) ions that can offer a wealth of information for peptide composition. However, MALDI-TOF/TOF is generally used for routine protein identification based on database search or de novo sequencing combined with chemical derivation. Consequently, the characteristics of IM ions may not be fully explored and utilized. Here, a total of 239 MS/MS spectra are used to explore the fragmentation features of IM ions with MALDI TOF/TOF spectrometry and their application for peptide identification. IM ion signals can be observed for 14 kinds of amino acids including histidine etc with a positive rate of more than 50%. We have found that the chemical nature of the amino acids and position effects are the two main factors that affect the intensity of fragment ions. In addition, false positive IM ions are mainly derived from Arg, Lys, Leu and Ile residues or mixture peptides. Besides the compositional information, partial sequence information can also be obtained by a comparison of the relative intensity of IM ions. These findings are helpful when performing manual interpretations and could be useful for improving current peptide search algorithms.%基质辅助激光解吸电离-串联飞行时间质谱( MALDI-TOF/TOF)产生的亚胺离子可以提供丰富的肽段组成信息，该方法通常用于基于数据库搜索的蛋白质鉴定，或者结合化学衍生法用于从头测序，因而在一定程度上限制了对亚胺离子的认识及应用。本研究利用239个串联质谱探索MALDI-TOF/TOF 中亚胺离子的断裂特征以及它们在肽段鉴定中的应用，发现在高能碰撞诱导解离条件下组氨酸等14种氨基酸可产生较强的亚胺离子信号(>50%阳性率)，氨基酸的化学结构、位置效应和氨基酸残基个数是影响碎片离子强度的主要因素。此外，探讨了亚胺离子应用过程中的假阳性问题，提出亚胺离子相对强度的比较可以降低假阳性和提高肽段鉴定确定度，有助于完善目前的数据库搜索算法和辅助从头测序分析。
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