Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins.
Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins.
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磷酸化对完整磷蛋白质谱定量的影响。
DOI：
10.1021/acs.analchem.7b05246
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发表时间：
2018
影响因子：
7.4
通讯作者：
Ge,Ying
中科院分区：
文献类型：
--
作者：
Wu,Zhijie;Tiambeng,TimothyN;Cai,Wenxuan;Chen,Bifan;Lin,Ziqing;Gregorich,ZacheryR;Ge,Ying
蛋白质磷酸化是一种普遍存在的重要的翻译后修饰（PTM），参与许多细胞过程。基于质谱（MS）的蛋白质组学已经成为蛋白质鉴定、表征和定量的优选技术。而在电喷雾电离（ESI）-MS中的肽的电离/检测效率显着影响的磷酸化的存在下，完整的蛋白质的物理化学性质被认为不会显着变化，由于相对较小的修改大的完整蛋白质。因此，假设完整磷蛋白的电离/检测效率对于后续MS定量没有明显改变。然而，这一假设从未得到严格的检验。在此，我们系统地研究了磷酸化对单磷酸化和多磷酸化蛋白质的ESI-MS定量的影响。我们证实，一个单一的磷酸化并没有明显影响ESI-MS定量磷蛋白的表现在谜同源异构体2（28 kDa）与单磷酸化。此外，不同的电离和去溶剂化参数不影响磷蛋白定量。与单磷酸化相反，多磷酸化显著影响磷蛋白的ESI-MS定量，这可能是由于未磷酸化和磷酸化蛋白质形式之间的差异电离/检测效率，如pentakis磷酸化β-酪蛋白（24 kDa）所示。
Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based proteomics has emerged as the preferred technology for protein identification, characterization, and quantification. Whereas ionization/detection efficiency of peptides in electrospray ionization (ESI)-MS are markedly influenced by the presence of phosphorylation, the physicochemical properties of intact proteins are assumed not to vary significantly due to the relatively smaller modification on large intact proteins. Thus, the ionization/detection efficiency of intact phosphoprotein is hypothesized not to alter appreciably for subsequent MS quantification. However, this hypothesis has never been rigorously tested. Herein, we systematically investigated the impact of phosphorylation on ESI-MS quantification of mono- and multiply phosphorylated proteins. We verified that a single phosphorylation did not appreciably affect the ESI-MS quantification of phosphoproteins as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation. Moreover, different ionization and desolvation parameters did not impact phosphoprotein quantification. In contrast to monophosphorylation, multiphosphorylation noticeably affected ESI-MS quantification of phosphoproteins likely due to differential ionization/detection efficiency between unphosphorylated and phosphorylated proteoforms as shown in the pentakis-phosphorylated β-casein (24 kDa).