Everley Robert A, Huttlin Edward L, Erickson Alison R, Beausoleil Sean A, Gygi Steven P
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Laboratory of Systems Pharmacology, Harvard Medical School , Boston, Massachusetts 02115 United States.
J Proteome Res. 2017 Feb 3;16(2):1069-1076. doi: 10.1021/acs.jproteome.6b00487. Epub 2016 Dec 30.
While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.
在开发一种多重磷酸酪氨酸肽定量分析方法时，我们有了一个意外发现：含磷酸酪氨酸（pY）的肽出现了显著的中性丢失。我们使用一个包含2000种肽的文库，通过使用高分辨率高质量精度的轨道阱仪器，在CID、HCD和ETD碎片化过程中，将未标记的肽与两种最高多重性的等压标签（iTRAQ8和TMT10）进行比较，试图系统地研究这一发现。我们发现pY肽的中性丢失行为与质子迁移率降低一致，并且在ETD过程中不会发生。由于TMT标记，肽N端的质子化位点从伯胺变为叔胺，这会增加该位点的气相碱性并降低质子迁移率。这种碎片化行为的变化在仪器方法开发和MS/MS谱图解释以及后续的跟踪研究中都有影响。我们展示了如何使用中性丢失和磷酸酪氨酸铵离子，将通过搜索和位点定位算法未定位到酪氨酸的位点可靠地重新分配给酪氨酸。我们相信这些发现对于那些使用等压标签研究pY信号转导的人来说将具有普遍的意义。
