作者将承认斯科特·霍华德（Scott Howard）和阿比盖尔·马里纳克（Abigail Marinack

所有涉及人体尿液样本的研究都遵循了Pharmasan / Neuroscience Institutional Review Board（IRB）批准的手术。 尿液样品收集和肌酐（Cr）测定<ol><li>第二天早晨收集5 mL尿样。 10小时过夜禁食在含有250μL3 N HCl作为防腐剂的5 mL输送管A中，储存于-20°C直至使用。 </li><li>解冻并涡旋5 mL运输尿A管和离心机离心机（ 如 Sorvall）（2297 xg，10分钟）。 </li><li>从5 mL运输管A等分1 mL尿液两次</li><li>通过尿肌酐法测定Cr 26 。 </li></ol> 2.准备标准，IS和质量控制（QC）样品<ol><li>在含有0.01％HCOOH流动相A（MA）的水中以100μg/ mL制备3-NT的储备溶液，并储存在-20℃。 </li><li>使通过用MA稀释100μg/ mL 3-NT储备溶液，浓度为100ng / mL的3-NT工作标准溶液。 </li><li>通过用不含3-NT的0.15M HCl酸化空白尿以及空白和双倍空白样品稀释100ng / mL工作标准，产生5至2500pg / mL的标准品。 </li><li>用MA在水中以100μg/ mL制备IS 13 C 9 -3-NT的储备溶液，并储存在-20℃。 </li><li>通过用MA稀释100μg/ mL 13 C 9 -3-NT IS储备溶液，使其成为500 pg / mL的IS工作溶液。 </li><li>通过在酸化的空白尿中稀释3-NT工作标准，建立覆盖LLOQ，低，中和高水平（ 即 10,25,100,5500和1,250pg / mL）的五个等级的QC样品。 </li></ol>固相萃取法<ol><li>解冻和涡流尿样，标准品和QC样品。 </li><li>添加尿样，标准品和QC（每个250μL）至清洁的2mL 96孔收集板的32个孔中。 </li><li>将500pg / mL IS工作溶液（50μL）引入每个孔，除了双空白样品孔。向双层样品孔中加入0.01％HCOOH（50μL）。 </li><li>加入0.1％HCOOH（250μL）的LC-MS / MS水。 </li><li>将上述混合物与8通道移液管混合三次。 </li><li>盖板，直到SPE加载。 </li><li>将MCX 96孔提取板和收集容器放在正压处理器上。 </li><li>通过吸附剂使流动的MeOH（200μL）对提取板进行调节。 </li><li>用2％HCOOH（200μL）通过吸附剂进行平衡。 </li><li>将每个预混合样品的整个体积用8通道移液管小心地装入预处理提取板上。 </li><li>在正压处理器上设置低正压（ 例如 3 psi），以使混合物流过缓慢吸收剂，如果需要，调整压力。 </li><li>用2％HCOOH（200μL）将水冲洗通过吸附剂清洗孔。 </li><li>通过使甲醇（200μL）通过吸附剂清洗孔。 </li><li>用水（200μL）通过吸附剂清洗孔。 </li><li>在正压处理器上以高正压设置（ 例如 40 psi）完全干燥油井。 </li><li>用干净的2 mL 96孔收集板更换储存器。 </li><li>应用pH 9（25μL）的25 mM NH 4 OAc，从萃取板中洗脱保留的分析物和IS。 </li><li>移取5％HCOOH（50μL）的LC-MS水中和洗脱液。 </li><li>与8通道移液管混合三次，并提交给LC-MS / MS站进行分析。 </li></ol> LC-MS / MS分析<ol><li>用量筒精确测量2,000 mL LC-MS水，并将其转移到2升瓶中。 </li><li> Pipette将200μL纯HCOOH装入含有LC-MS水的上述瓶中。 </li><li>充分混合并标记为流动相A（MA）。包括缩写，准备日期和到期日。 </li><li>取2升瓶的LC-MS甲醇，标记为流动相B（MB），开始日期和有效期。 </li><li>将自动进样器的温度设置为4°C。 </li><li>将带有准备样品的收集板放入自动进样器。 </li><li>将PFP柱（150 mm x 2.1 mm id，3μm）和保护柱放在烤箱中。 </li><li>将烤箱温度设置为30°C。 </li><li>使用采集方法用LC梯度洗脱平衡10分钟，如表1所示。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时间（分钟） </td><td> 模 </td><td> 活动 </td><td> 参数 </td></tr><tr><td> 0 </td><td>水泵</td><td>泵B浓度</td><td>五</td></tr><tr><td> 0.5 </td><td>水泵</td><td>泵B浓度</td><td> 20 </td></tr><tr><td> 1 </td><td>水泵</td><td>泵B浓度</td><td> 50 </td></tr><tr><td> 3 </td><td>水泵</td><td>泵B浓度</td><td> 80 </td></tr><tr><td> 4 </td><td>水泵</td><td>泵B浓度</td><td> 90 </td></tr><tr><td> 4.01 </td><td>水泵</td><td>泵B浓度</td><td> 95 </td></tr><tr><td> 5.5 </td><td>水泵</td><td>泵B浓度</td><td> 95 </td></tr><tr><td> 5.6 </td><td>水泵</td><td>泵B浓度</td><td>五</td></tr><tr><td> 7 </td><td>调节器</td><td>停止</td><td></td></tr></tbody></table> 表1: 液相色谱梯度洗脱条件 <ol start="10"><li>创建一个批处理列表，其中包括标准品，QC和尿样。 </li><li>通过注射准备的样品（12μL）开始批次。 </li></ol>峰值识别，集成和数据处理<ol><li>使用该软件控制数据采集和处理。 </li><li>识别并整合所有样品的3-NT和IS峰。 </li><li>通过3-NT和IS与标称3-NT浓度的峰面积比以1 / x的线性回归线性回归，建立10-2,500pg / mL范围的3-NT定量的标准曲线。 </li><li>使用标准曲线量化所有样品。 </li><li>确定QC样品是否落在确定的范围内。 </li><li>将检测到的尿样浓度以nM或nmol / mmol Cr为单位转换为最终结果。 </li></ol>

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作者宣称他们没有利益冲突。

在浓度实质变化先前在文献中内源性游离3-NT报导在人尿样品揭示了与可用的测定8，9，10，11相关联的方法问题。准确测定人尿中3-NT的低基础水平仍然是一项具有挑战性的任务，需要对样品制备和LC-MS / MS分析采取特殊预防措施。该协议概述了一种新型SPE程序，结合选择性LC-MS / MS检测，允许以高通量对...

氧化应激的临床表现的影响已被推到前列，近年来1。正在探索的生物标志物之一是3-硝基酪氨酸（3-NT），当活性氮物质（RNS）与酪氨酸，儿茶酚胺神经递质前体相互作用时形成最终稳定的产物。而3-NT可以具有临床价值作为体内 RNS的生物标志物，其属性和酪氨酸的功能的显着变化可能不利地相应的蛋白质和细胞功能1,2影响。新兴的研究表明，3-NT可以在炎症条件下发挥重要作用3，神经退行性疾病4，5，心血管疾病6和7糖尿病，以及与氧化应激的条件。但是，这些都是这样的rvations基于来自缺乏灵敏度和/或选择性8，9，10，11的方法的结果。以前在文献中报道的生物样品的3-NT浓度范围巨大，显示出严重的分析问题与这些检测有关，需要进行技术改进来准确量化3-NT的水平，并验证其在这些病症的病理学中的作用。 免费3-NT生物基质的定量呈现人与仪器8，9，10，11一个特殊的挑战。首先，内源性3-NT的痕量水平要求超敏感检测;第二，存在众多结构相似的类似物，特别是酪氨酸大量过剩，需要高度的选择性;第三，通过酪氨酸硝化与无处不在的硝酸盐和亚硝酸盐的3-NT的人工形成需要在样品制备过程中特别考虑，以避免过度估计3-NT。 中的各种各样的用于测量3-NT方法的，MS / MS一直被认为是金标准方法由于其优越的灵敏度和选择性11，12，13，14。气相色谱（GC），其耦合MS / MS提供最佳的灵敏度，然而，必不可少的样品衍生步骤过于繁琐和耗时是有效的临床效用15,16。 LC-MS / MS不需要复杂的样品衍生化，使其成为更有希望的选择。尽管如此，还有几个要克服的障碍的文献中报道的LC-MS / MS方法nsitivity需要改善的测量低丰度3-NT 7，17，18和相对长的处理时间必须缩短用于高通量应用12，13，17，19 。 另外，在考虑临床应用时，所用的生物矩阵起着重要的作用。它应该是容易且廉价获得和非侵入性的如果可能的话20，21，22。等离子体是文献中传统使用的样品，不是临床上理想的基质，因此寻求利用非侵入性和成本效益的尿液的方法。 几个尝试发展reliable和特定LC-MS / MS方法已经使用尿9，10，11制成。然而，它们的选择性，可靠性或效率都不足以临床使用。已经提出使用传统反相柱（C18型）作为3-NT分析的样品清洗的主要SPE的有效性，并提出了强阳离子交换（SCX）和反相C18-OH的顺序SPE 6 ， 7，19。最近开发的LC-MS / MS方法采用手动C18 SPE，制备型高压液相色谱（HPLC）和在线SPE的多步纯化方法，用于3-NT 23的分析。尽管这种方法对于临床目的来说足够敏感，LLOQ为0.041nM，但是清除过程是密集和繁琐的，红色3 mL尿液，限制了其高通量的可行性。采用分子印迹聚合物作为SPE吸附剂，以提高清洗过程14的效率，但是所得到的LLOQ（0.7μg/ mL）对于临床标本不够低。另一种方法需要二维（2D）LC-MS / MS和免疫亲和层析来进行样品净化，以达到0.022nM 24的检测限（LOD）。虽然所有这些方法在3-NT的评估方面取得了进步，但没有一个已经实现了临床应用所需的灵敏度，可靠性和效率。 为了研究的自由3-NT病理和其在临床环境中的氧化应激的生物标志物的作用，我们已经开发出一种方法，它是简单，高效，准确，精确，可以实现高通量临床应用25。小型化混合模式阳离子实施了hange（MCX）96孔提取微孔板，以实现单次提取中简单有效的3-NT样品清除和富集，绕过了需要衍生化，蒸发和2D-LC的现有方法中看到的缺点。具有0.01％HCOOH作为流动相添加剂的液相色谱提供了快速循环时间的增强的信号响应。通过使用温和的NH 4 OAc洗脱溶液用于3-NT的选择性洗脱以及使用3-NT和内标（IS）的MRM转换，进一步改善了选择性。通过使用减少量的优选13 C-标记的同位素IS进行定量来补偿基质效应。随着这种方法的出现，研究人员和临床医生将能够验证3-NT在临床条件下的作用，并进一步探索氧化应激的影响。

<table><tbody><tr><td>3-Nitro-L-tyrosine</td><td>Sigma</td><td>N7389-5g</td><td></td></tr><tr><td>3-Nitro-L-tyrosine-13C&lt;sub&gt;9&lt;/sub&gt;</td><td>Sigma</td><td>652296-5.0mg</td><td></td></tr><tr><td>Mass Spec Gold Urine</td><td>Golden West Biologicals</td><td>MSG 5000-1L</td><td></td></tr><tr><td>Oasis MCX 96-well &amp;micro;Elution plate</td><td>Waters</td><td>186001830BA</td><td></td></tr><tr><td>2 mL 96 well collection plate</td><td>Phenomenex&amp;nbsp;&amp;nbsp;</td><td>AH0-7194</td><td></td></tr><tr><td>96 positive processor</td><td>Waters&amp;nbsp;</td><td>186005521</td><td></td></tr><tr><td>LC-MS Ultra CHROMASOLV methanol&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>14262-2L</td><td></td></tr><tr><td>LC-MS Ultra CHROMASOLV water</td><td>Sigma</td><td>14263-2L</td><td></td></tr><tr><td>Formic acid for mass spectrometry</td><td>Sigma</td><td>94318-50ML-F</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma</td><td>338818-1L</td><td></td></tr><tr><td>Ultra PFP propyl columns</td><td>Restek</td><td>9179362</td><td></td></tr><tr><td>5500 Triple quad</td><td>AB Sciex&amp;nbsp;</td><td>/</td><td>Contact manufacture for more detail</td></tr><tr><td>UFLC-XR</td><td>Shimadzu&amp;nbsp;</td><td>/</td><td>Contact manufacture for more detail</td></tr><tr><td>Integra 400 Plus&amp;nbsp;</td><td>Roche</td><td>/</td><td>Urinary Creatinine Jaff&amp;eacute; Gen 2 method</td></tr><tr><td>LCMS certified 12 x 32 mm screw neck vial</td><td>Waters</td><td>600000751CV</td><td></td></tr><tr><td>LCGC certified 12 x 32 mm screw neck total recovery vial</td><td>Waters</td><td>186000384C</td><td></td></tr><tr><td>5 mL transport tube</td><td>Phenix</td><td>TT-3205</td><td></td></tr><tr><td>50 mL Centrifuge tube</td><td>Crystalgen&amp;nbsp;</td><td>23-2263</td><td></td></tr><tr><td>15 mL Centrifuge tube</td><td>Crystalgen&amp;nbsp;</td><td>23-2266</td><td></td></tr><tr><td>eLine electronic pipette</td><td>Sartorius</td><td>730391</td><td></td></tr><tr><td>Microfuge centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>A46474</td><td></td></tr><tr><td>OHAUS balance&amp;nbsp;&amp;nbsp;</td><td>Kennedy Scales, inc.</td><td>735</td><td></td></tr><tr><td>Vortex mixer&amp;nbsp;</td><td>Bernstead Thermolyne</td><td>M16715</td><td></td></tr></tbody></table>

integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications

游离的3-硝基酪氨酸（3-NT）已广泛用作氧化应激的可能生物标志物。在各种病理状况下报道了3-NT的水平升高。然而，现有方法缺乏足够的灵敏度和/或特异性，可靠地测量3-NT的低内源水平，对于临床应用来说太麻烦了。因此，迫切需要进行分析改进，以准确量化3-NT的水平，并验证3-NT在病理状况中的作用。该方案介绍了新型液相色谱串联质谱（LC-MS / MS）检测与小型化固相萃取（SPE）的开发，用于快速准确测量人尿中3-NT作为非侵入性生物标志物用于氧化应激。使用96孔板的SPE通过组合样品净化和分析物富集显着简化了工艺，而无需繁琐的衍生化和蒸发步骤，减少溶剂消耗，废物处理，污染风险和整体处理时间。使用pH为9的25mM乙酸铵（NH 4 OAc）作为SPE洗脱溶液，显着提高了选择性。通过调整多重反应监测（MRM）转换，改善了质谱信号的响应。在五氟苯基（PFP）柱（150mm×2.1mm，3μm）上使用0.01％HCOOH作为添加剂将信号响应提高2.5倍，并将总运行时间缩短至7分钟。实现了10 pg / mL（0.044 nM）的定量下限（LLOQ），相对于所报告的测定显示出显着的灵敏度改善。这种简化，快速，选择性和敏感性方法允许在24小时的时间段内处理两个尿液样本（n = 192）。考虑到明显改善的分析性能和非侵入性和便宜的尿液采样，所提出的检测方法有利于临床前和临床学习。

图1说明，在优化的LC条件下，3-NT与其它结构相似的酪氨酸类似物完全色谱分离，这消除了由于这些极度过量的化合物引起的共洗脱干扰，从而提高了测定选择性的程度。此外，以0.01％HCOOH作为添加剂在MA和甲醇中的流动速率为0.45 mL / min的梯度洗脱可以快速洗脱3-NT（ 即 3分钟，周转时间为7分钟）。 <p class="jove_content" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications at JoVE.com

微型固相萃取与 LC-MS/MS 检测人尿液中 3-硝基酪氨酸的集成在临床应用中的应用化学|视频

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

开发了选择性和灵敏的液相色谱串联质谱（LC-MS / MS）方法与混合模式阳离子交换（MCX）96孔微量培养板上的有效固相萃取相结合，用于测量游离的3-硝基酪氨酸（ 3-NT），具有高通量，适用于临床应用。

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

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Osmoregulation and Excretion

SCIENTISTS IN ACTION

9.2K 次观看.  Pharmasan Labs, Inc.. 开发了选择性和灵敏的液相色谱串联质谱（LC-MS / MS）方法与混合模式阳离子交换（MCX）96孔微量培养板上的有效固相萃取相结合，用于测量游离的3-硝基酪氨酸（ 3-NT），具有高通量，适用于临床应用。 

视频: Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

用细胞色素 P450s 检测亚硝胺的体外Nitrosamide 形成的一般方法

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

癌症研究

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

采样使用超探头棒棒糖样的土著人的唾液多肽：简化和增强肽的临床质谱检测

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

医学

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

利用高效液相色谱-电化学检测仪检测大气环境中的 3-硝基酪氨酸

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

环境科学

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

生物化学

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

蛋白质的MS检测快速酶法处理与流通型微反应器

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

生物工程

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...