MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments,Journal of Proteome Research

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MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-02-26 00:00:00 , DOI: 10.1021/acs.jproteome.8b00006
Matthew J. Berberich ₁ , Joao A. Paulo ₂ , Robert A. Everley _{1,

2}

Affiliation

Protein phosphorylation is critically important for many cellular processes, including progression through the cell cycle, cellular metabolism, and differentiation. Isobaric labeling, for example, tandem mass tags (TMT), in phosphoproteomics workflows enables both relative and absolute quantitation of these phosphorylation events. Traditional TMT workflows identify peptides using fragment ions at the MS2 level and quantify reporter ions at the MS3 level. However, in addition to the TMT reporter ions, MS3 spectra also include fragment ions that can be used to identify peptides. Here we describe using MS3 spectra for both phosphopeptide identification and quantification, a process that we term MS3-IDQ. To maximize quantified phosphopeptides, we optimize several instrument parameters, including the modality of mass analyzer (i.e., ion trap or Orbitrap), MS2 automatic gain control (AGC), and MS3 normalized collision energy (NCE), to achieve the best balance of identified and quantified peptides. Our optimized MS3-IDQ method included the following parameters for the MS3 scan: NCE = 37.5 and AGC target = 1.5 × 10⁵, and scan range = 100–2000. Data from the MS3 scan were complementary to those of the MS2 scan, and the combination of these scans can increase phosphoproteome coverage by >50%, thereby yielding a greater number of quantified and accurately localized phosphopeptides.

中文翻译：

MS3-IDQ：利用MS3光谱超出定量，在等压标记实验中增加了磷酸化蛋白质组的覆盖率

蛋白质磷酸化对于许多细胞过程至关重要，包括整个细胞周期，细胞代谢和分化过程。磷酸蛋白质组学工作流程中的等压标记，例如串联质量标签（TMT），可以对这些磷酸化事件进行相对和绝对定量。传统的TMT工作流程使用MS2级别的碎片离子识别多肽，并在MS3级别量化报告离子。但是，除了TMT报告离子外，MS3光谱还包括可用于识别肽的碎片离子。在这里，我们描述了将MS3光谱用于磷酸肽的鉴定和定量，我们将其称为MS3-IDQ。为了最大化量化的磷酸肽，我们优化了几个仪器参数，包括质量分析仪的模式（即离子阱或Orbitrap），MS2自动增益控制（AGC）和MS3归一化碰撞能量（NCE），以实现已鉴定和定量肽段的最佳平衡。我们优化的MS3-IDQ方法包括以下用于MS3扫描的参数：NCE = 37.5和AGC目标= 1.5×10⁵，扫描范围= 100–2000。来自MS3扫描的数据与MS2扫描的数据是互补的，这些扫描的组合可以使磷酸化蛋白质组覆盖率增加> 50％，从而产生更多数量的定量和精确定位的磷酸肽。

更新日期：2018-02-27

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