Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.
Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.
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DOI：
10.1002/rcm.7063
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发表时间：
2014-12-30
影响因子：
2
通讯作者：
Chang, Emmanuel J.
中科院分区：
文献类型：
--
作者：
Ho, Hsin-Pin;Rathod, Pratikkumar;Louis, Marissa;Tada, Christine K.;Rahaman, Sherida;Mark, Kevin J.;Leng, Jin;Dana, Dibyendu;Kumar, Sanjai;Lichterfeld, Mathias;Chang, Emmanuel J.
基质辅助激光解吸/电离（MALDI）质谱结合同位素标记方法是有效的蛋白质和肽定量，但在其复用能力，成本效益和动态范围有限。本研究探讨了MALDI MS为基础的量化肽磷酸化没有标记，并旨在克服拍摄到拍摄的MALDI变异性使用的数学变换和延长的数据采集时间。磷酸肽摩尔分数的倒数和磷酸化的信号比的倒数之间的线性关系的推导，并使用三个单独的磷酸肽系统含有磷酸化的丝氨酸，苏氨酸和酪氨酸残基的实验评估：混合物的磷酸肽和它的des-phospho-类似物与已知的化学计量测量真空MALDI-线性离子阱质谱和拟合的线性模型。该模型被验证用于定量体外磷酸化测定与Cdk 2/cyclinA的抑制研究。动态范围为皮摩尔至飞摩尔，具有良好的准确度（与预期值的偏差为1.5-3.0%）和重现性（RSD = 4.3-6.3%）。评价了经典抑制剂奥洛莫辛和r-roscovitine对细胞周期蛋白依赖性激酶磷酸化的抑制作用，发现IC 50值与文献报道的值一致。这些结果，实现了单点校准，没有同位素或色谱，比较有利的那些达到使用同位素稀释（p > 0.5的准确性）。这里推导出的数学关系可以应用于一种方法，我们称之为双倒数无同位素磷酸肽定量（DRIP-Q），作为体外磷酸化测定的定量策略，这是第一种基于MALDI的无同位素和校准曲线的方法。这些结果也为进一步系统研究肽组成和实验条件对定量、无标记MALDI的影响铺平了道路。
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with isotope labeling methods are effective for protein and peptide quantification, but limited in their multiplexing capacity, cost-effectiveness and dynamic range. This study investigates MALDI MS-based quantification of peptide phosphorylation without labeling, and aims to overcome the shot-to-shot variability of MALDI using a mathematical transformation and extended data acquisition times. A linear relationship between the reciprocal of phosphopeptide mole fraction and the reciprocal of phosphorylated-to-unphosphorylated signal ratio is derived, and evaluated experimentally using three separate phosphopeptide systems containing phosphorylated serine, threonine and tyrosine residues: mixtures of phosphopeptide and its des-phospho-analog with known stoichiometry measured by vacuum MALDI-linear ion trap mass spectrometry and fit to the linear model. The model is validated for quantifying in vitro phosphorylation assays with inhibition studies on Cdk2/cyclinA. Dynamic range of picomoles to femtomoles, good accuracy (deviations of 1.5–3.0% from expected values) and reproducibility (RSD = 4.3–6.3%) are achieved. Inhibition of Cyclin-dependent Kinase phosphorylation by the classical inhibitors olomoucine and r-roscovitine was evaluated and IC50 values found to be in agreement with reported literature values. These results, achieved with single-point calibration, without isotope or chromatography, compare favorably to those arrived at using isotope dilution (p > 0.5 for accuracy). The mathematical relationship derived here can be applied to a method that we term Double Reciprocal Isotope-free Phosphopeptide Quantification (DRIP-Q), as a strategy for quantification of in vitro phosphorylation assays, the first MALDI-based, isotope- and calibration curve-free method of its type. These results also pave the way for further systematic studies investigating the effect of peptide composition and experimental conditions on quantitative, label-free MALDI.
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