MS/MS/MS reveals false positive identification of histone serine methylation.
MS/MS/MS reveals false positive identification of histone serine methylation.
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MS/MS/MS 揭示了组蛋白丝氨酸甲基化的假阳性鉴定。
DOI：
10.1021/pr900864s
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发表时间：
2010
影响因子：
4.4
通讯作者：
Zhao,Yingming
中科院分区：
文献类型：
--
作者：
Zhang,Junmei;Chen,Yue;Zhang,Zhihong;Xing,Gang;Wysocka,Joanna;Zhao,Yingming
赖氨酸和精氨酸残基的甲基化在调节组蛋白的结构和功能中起着关键作用。然而，组蛋白中其他氨基酸残基的甲基化以前还没有被描述过。利用穷尽的纳米高效液相色谱/质谱仪和盲法蛋白质序列数据库检索，我们初步确定了小牛胸腺组蛋白H3丝氨酸28的甲基化状态。这一分配符合我们严格的手动验证规则、高效液相/质谱仪/质谱仪中的洗脱和相应的合成肽、不同细胞系中这种甲基化的动态性质以及同位素标记。然而，仔细检查了一系列合成肽的MS/MS和MS/MS/MS光谱，证实甲基化实际上发生在K27而不是S28。错误的分配是因为推定的S28甲基化多肽的(y9+14)和K27甲基化多肽的(b9+18)离子共享Samem/z值(m/z801)。这一MS/MS峰被用作确定S28(连续的Y8和(Y9+14)离子)甲基化的主要证据。MS/MS/MS分析揭示了丝氨酸甲基化的假阳性性质：含糊的离子ATM/z801确实是(b9+18)，是碰撞激活解离(CAD)过程中气相Anin玻璃化反应产生的离子。当赖氨酸(K27)被乙酰化时，苏钦的体外反应程度大大降低，当C末端被羧基衍生化封闭时，这种反应完全消除。此外，这种侧链辅助的C-末端重排是电荷依赖的。综上所述，这些结果表明，在解释翻译后修饰(PTM)数据时应格外小心，并且需要合成多肽的MS/MS和MS/MS/MS来验证含有新PTM的多肽的身份。
Methylation of lysine and arginine residues is known to play a key role in regulating histone structure and function. However, methylation of other amino acid residues in histones has not been previously described. Using exhaustive nano-HPLC/MS/MS and blind protein sequence database searches, we tentatively assigned methylation to serine 28 of histone H3 from calf thymus. The assignment was in agreement with our stringent manual verification rules, coelution in HPLC/MS/MS with its corresponding synthetic peptide, the dynamic nature of such methylation in distinct cell lines, and isotopic labeling. However, careful inspection of the MS/MS and MS/MS/MS spectra of a series of synthetic peptides confirmed that methylation actually occurs on K27 rather than on S28. The misassignment was caused by the fact that the (y9+ 14) of the putative S28-methylated peptide and (b9+ 18) ions of the K27 methylated peptide share the samem/zvalue (m/z801). This MS/MS peak was used as the major evidence to assign methylation to S28 (consecutive y8and (y9+ 14) ions). MS/MS/MS analysis revealed the false positive nature of serine methylation: the ambiguous ion atm/z801 is indeed (b9+ 18), an ion resulting from anin vitroreaction in the gas phase during collisionally activated dissociation (CAD). When lysine (K27) was acetylated, the degree of suchin vitroreactions was greatly reduced, and such reactions were completely eliminated when the C-terminus was blocked by carboxylic group derivatization. Moreover, such side-chain assisted C-terminal rearrangement was found to be charge dependent. In aggregate, these results suggest that extra caution should be taken in interpretation of post-translational modification (PTM) data and that MS/MS as well as MS/MS/MS of synthetic peptides are needed for verifying the identity of peptides bearing a novel PTM.