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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis
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Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

机译：基于纳米金刚石的多步和单步磷酸化肽的两步采样，用于MALDI-TOF质谱分析

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摘要

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO _2-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1:1000, which out-performs the commercial Fe~(3+)-IMAC and TiO_2 isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.
机译：由于电离过程中的抑制作用，使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）同时检测多种和单磷酸化的肽具有挑战性。为了克服这个问题，本研究提出了一种新方法，可通过使用聚精氨酸涂层（PA涂层）和二氧化钛涂层（TiO _2涂层）纳米金刚石进行逐步富集来改进磷酸肽的检测，以分离倍增和单磷酸化探针进行MALDI MS分析前先测定多肽。使用包含不同数量的磷酸基团的合成肽，α-酪蛋白，β-酪蛋白的胰蛋白酶消化物和复杂的蛋白质混合物证明了这种方法的可行性。该方法具有很高的特异性，它以低至1：1000的摩尔比有效地富集和分级分离了β-酪蛋白和牛血清白蛋白消化物中的磷酸肽，胜过了商用的Fe〜（3+） -IMAC和TiO_2隔离套件。它为通过MALDI MS分离和鉴定多重磷酸化和单个磷酸化的肽提供了一种简单有效的替代方法，并允许从有限的原料中推断出更多信息。

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