作者确认了由国家科学基金会资助的智能材料设计、分析和加工财团 (SMATDAP), IIA-1430280 合作协议, 以及研究生奖学金的 LA 董事会。这些实验的聚合物样品是由 MilliporeSigma (西格玛-爱秩序) 提供的。 本文的开放访问发布由 MilliporeSigma 赞助。

注意: 所有反应都是在通风罩中运行的。请阅读所有材料安全数据表 (MSDS) 的任何化学品使用, 并采取适当的预防措施。
1. 样品准备
<ol><li>基质库存溶液的制备<ol>
<li>将20毫克的α-氰基-4-肉桂酸 (HCCA) 溶于1毫升的四氢呋喃-unstabilized (THF), 直至溶解。</li>
		<li>将20毫克的 25-苯甲酸酸 (DHB) 溶解在1毫升的 THF 中, 并在漩涡中溶解。</li>
	</ol>
</li>
	<li>碱阳离子砧木溶液的制备<ol>
<li>将2毫克的三氟乙酸钠 (NaTFA) 溶于1毫升 THF, 直至溶解。</li>
		<li>将2毫克三氟乙酸钾 (KTFA) 溶于1毫升 THF, 直至溶解。</li>
	</ol>
</li>
	<li>分析物库存溶液的制备<ol>
<li>样品 1: 在0.5 毫升的 THF 中溶解2毫克的聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000), 直到溶解。</li>
		<li>样品 2: 在0.5 毫升的 THF 中溶解2毫克的聚氧乙烯二 (叠氮化物) (Mn = 2000), 涡旋直至溶解。</li>
		<li>样品 3: 溶解2毫克聚 (l-丙交酯), 硫醇终止 (Mn = 2500) 在0.5 毫升的 THF, 和漩涡, 直到溶解。</li>
	</ol>
</li>
	<li>样品混合物的制备分析<ol>
<li>通过混合基质、分析物和阳离子溶液来制备一系列溶液, 同时改变组分的相对比例, 使九种独特的样品混合。例如, 保持添加的矩阵库存解决方案常量的数量 (e. g., 10 µL), 用三 (e.、45、15和5µL) 的因子来改变分析物溶液的数量, 同时也改变阳离子溶液的用量, 其系数为三 (e。., 9, 3 和1µL)。这些样本有效地产生了 x 和 y 轴上两个不同浓度变量的 3 x 3 格样本 (图 1)。</li>
		<li>对于代表性的例子, 结合15µL 的聚 (l-丙交酯) 溶液, 15 µL 的 DHB 溶液, 1 µL NaTFA 溶液。</li>
		<li>将每个溶液混合物的 1 ul 在 MALDI 靶板上的单个试样上 (图 2)。在小的部分中以增量方式添加样本, 以防止样品从样品中流出, 使每个整除在添加额外样品之前蒸发干燥。 注: 对于更高沸点的溶剂, 可能需要用气枪来加速溶剂蒸发, 但应谨慎避免加热样品板, 这可能会导致板材变形。</li>
	</ol>
</li>
	<li>校准用标准样品的制备<ol>
<li>使用建议的供应商协议准备校准标准。 注: 大分子 calibrants 为本研究选择, 可作为纯大分子或预混与基质, calibrant 和阳离子在优化比率。</li>
	</ol>
</li>
</ol>2. 数据采集优化
<ol><li>启动数据采集<ol>
<li>开放式数据采集软件 "FlexControl"。</li>
		<li>通过按 "<img alt="Equation 1" src="/files/ftp_upload/57174/57174eq1v2.jpg">" 按钮, 弹出平台以启用目标板的加载。</li>
		<li>在适当的方向上, 轻轻地将靶板与加载的 calibrant 和分析物样品放在平台上。</li>
		<li>使用采集软件将目标板插入平台上, 再按 "<img alt="Equation 1" src="/files/ftp_upload/57174/57174eq1v2.jpg">" 按钮。</li>
		<li>通过按下文件选择适当的数据采集方法 (正模式采集方法) |选择方法。 注: 对于大多数聚合物样品, 包括我们的代表性分析物, 电离是期望通过与阳离子络合, 因此, 正模式获取方法是最合适的。根据仪器, 为更低的质量范围 (500-10,000 Da) 或当需要更高的分辨率时, 选择反射器模式方法文件。对于更高的分子量样本, 或者当需要较高的信号灵敏度时, 可接受较低分辨率, 选择线性模式方法文件。</li>
		<li>在获取数据之前, 请确保选择了收集数据的适当质量范围-理想情况下, 质量范围将包括预期分布中最低质量的一半, 以及预期分布中的最大质量。通过单击 "检测" 选项卡并查看质量范围来检查此问题。 注意: 这有助于确保样本中可能存在的低分子量降解片段或更高分子量聚集 (二聚体) 的信号包含在数据集中。另外, 请注意, 在大多数 MALDI 的质量谱中, 矩阵低聚物经常被注意到高信号强度, 提供高强度的噪声与质量高达 1000 Da, 复杂的分析下面的质量。虽然在获得最终数据集之前需要进行校准, 但只有当使用相同的采集参数作为为特定分析物进行优化的条件时, 才能获得精确的校准文件。因此, 在校准前需要对分析物质谱进行初步优化, 其次是校准的分析物质谱的重新取得。</li>
	</ol>
</li>
	<li>初步数据采集<ol>
<li>从采集软件中, 选择与所需分析物对应的目标板上的位置。</li>
		<li>启动收集数据, 同时移动的激光目标周围的样本, 以最大的信号。要启动数据收集, 请按 "开始"。 注: 激光在重复取样后可在特定位置排出基体。</li>
		<li>使用相机窗口左侧的滑动条, 调整激光功率, 实现同位素分辨率所需的最小功率。 注: 在分析多个样品以确认所研究的测定物、阳离子和基体的最佳比值时, 在每个分析样品上使用相同的激光功率, 以确定哪个样品对这些采集参数具有最佳的信噪比。继续未来的收购优化与样品, 似乎表现出最佳的信噪比。</li>
	</ol>
</li>
	<li>数据采集优化<ol>
<li>放大在一个单独的峰值在大范围的兴趣, 通过调整加速度电压的差异来优化分辨率 (对于本研究中的仪器, 这涉及到调整 "IS2" 值), 这是在光谱仪选项卡。 注意: 这是最快速优化通过改变 IS2 值在大的步骤, 注意到哪个值产生最佳分辨率 (i., 最小的全峰值宽度在一半最大信号强度), 然后进一步优化的小步骤IS2 值。通常情况下, 低质量聚合物的最佳 IS2 值较高 (接近 IS1), 而高质量聚合物则较低。</li>
		<li>如果需要, 请使用 reflectron 模式增加分辨率。 注: Reflectron 模式允许对同一m- /z 的离子的初始速度的差异进行补偿, 方法是将同一m-/z值的较高速度离子强制为探测器的较长路径。该探测器路径的增加使得相同的m-/z值的慢离子同时到达探测器, 有效地聚焦离子以提高分辨率。虽然 reflectron 模式通常改善信号分辨率, 对于弱信号强度的样本, 为了可视化数据, 可能需要线性模式。</li>
		<li>最后, 通过尽可能低的激光功率来优化激光功率, 同时还能产生合理的信噪比 (e., 信噪比约为 10)。 注: 由于较高的激光功率通常会降低分辨率, 并能诱发碎片, 因此使用激光能量减少而获得的质量谱质量最好, 但扫描次数更高。</li>
		<li>当采集参数优化后, 通过选择文件保存无标定质量谱, 然后将频谱保存为文件。对于外部校准, 在进行新的采集以生成分析物的校准质量谱之前, 必须在这些相同的、优化的参数下进行新的 calibrant 获取。</li>
	</ol>
</li>
</ol>3. MALDI 校准
<ol><li>校准质量谱的获取<ol>
<li>利用已为分析物优化的采集参数, 获得质量标准样本的最佳质量谱。 注: 理想情况下, 校准集应该包括一个标准高于兴趣范围, 一个低于, 至少一个在兴趣范围内。如果两个样品的所有采集参数都相同, 则校准的准确度最好。</li>
	</ol>
</li>
	<li>创建校准文件<ol>
<li>确保任何现有的校准是无效的或在一个位置被压制无效校准下的光谱仪标签。</li>
		<li>使用相同的采集参数 (例如, 激光功率, IS2 电压), 通过选择与光标对应的井, 将激光移到含有 calibrant (大分子标准、肽) 的样品井中, 并获得一个按开始的频谱。</li>
	</ol>
</li>
	<li>一旦获得足够的信号, 按开始完成获取数据。</li>
	<li>一旦获得了 calibrant 的质量频谱, 请在对应于该校准标准的校准选项卡中选择 "质量控制列表" 下拉菜单。适当的质量控制名单将有选择的 calibrant 的参考大量以适当的阳离子。 注: 这些应由 calibrant 供应商提供, 并确保在达到同位素分辨率时使用 monoisotopic 质量值 (e., 在m/z以下的反射器模式 = 5000), 以及当同位素分辨率时的平均质量值无法实现 (例如, 在m/z以上的线性模式 = 5000),</li>
	<li>在将相应的参考峰值与每个选定的 calibrant 峰值匹配之前, 请确保选择 "处理" 选项卡使用适当的峰值领料协议。 注意: 峰值拾取协议可以根据光谱分辨率而变化。对于平均质量计算, 该软件应采取的质量平均值横跨整个系列的同位素峰值。对于 monoisotopic 质量计算, 应将该软件设置为仅计算第一同位素峰值的精确质量。</li>
	<li>将参考质量从质量控制列表应用到相应的 calibrant 质量谱信号, 方法是选择感兴趣峰值左侧的区域, 然后单击要应用的控制列表中相应的质量。继续处理剩余的 calibrant 峰值。 注: 对于最精确精确的校准, 将分析物和 calibrant 样品尽可能紧密地放在目标板上, 因为目标板高度的细微变化会影响校准的准确性。</li>
	<li>对优化后的采集参数进行质量尺度标定后, 重新获得分析谱。</li>
</ol>4. 数据分析和解释
<ol><li>峰值采摘<ol>
<li>在数据分析软件 (FlexAnalysis) 中打开解析物谱。</li>
		<li>放大在一个峰值, 以确定是否同位素分辨率已达到通过选择缩放 X 范围按钮。</li>
		<li>新闻质量列表 |查找选择峰值。如果 monoisotopic 峰得到解决, 选择这第一峰值在同位素分布, 以确定其质量使用一个 monoisotopic 的峰值采摘协议。如果 monoisotopic 峰值未得到解决, 则使用平均质量峰值拾取协议, 并确定整个同位素分布的平均质量。</li>
		<li>继续这个峰值采摘过程中的每个 n 在聚合物分布。</li>
	</ol>
</li>
	<li>聚合物表征与端组分析计算 注: 正确使用时, MALDI 可为聚合物的质量分布计算提供有价值、准确的数据。应该指出的是, 当聚合物样品的分散性相对较低 (例如, 大约Đ= 1.3 或以下) 时, 质量分布数据才准确。<ol>
<li>用公式计算每个质量分数的摩尔数的平均分子量、质量平均值: <img alt="Equation 2" src="/files/ftp_upload/57174/57174eq2v2.jpg"> 其中Ni = 特定分子量的分子数量和Mi = 这些分子的特定分子量。</li>
		<li>计算重量平均分子量, 质量平均值与每个质量分数的重量, 使用公式: <img alt="Equation 3" src="/files/ftp_upload/57174/57174eq3v2.jpg"> 其中 N i = 特定分子量的分子数量和 M i = 这些分子的特定分子量。</li>
		<li>一旦计算了 mw和 mn , 就用Đn的比值来量化分子量分布的广度, 即"分散性"。</li>
		<li>MALDI MS 数据分析的最独特和最强大的特点是能够确定或确认均聚物的最终组。通过重新排列在质量谱 (M) 中观测到的 n 的质量, 确定最终组: m = n (EG1 ) + m EG2 + m离子 其中n = 聚合的程度, MEG1 = 质量α末端小组, MEG2 = 质量ω末端小组, M = 聚合物的重复单元的质量, 和M离子 = 质量的离子那配合与聚合物。</li>
	</ol>
</li>
</ol>

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</ol>

作者对本研究中使用的球形 calibrants 有一定的经济利益。

MALDI 质谱是一种无价的聚合物表征分析工具, 因为它能够在单一带电的状态下产生聚合物离子, 并具有极小的碎片。这种软电离技术利用短激光脉冲对嵌入在基质化合物中的聚合物分析物的固态样品脱附在气相中产生聚合物离子。大分子通常是通过与阳离子络合而成的, 加入到基质中, 以质谱法进行分析。然后通过萃取电压加速这些大分子离子, 使它们进入飞行管的无场区域, 从而使它们的m-/z根据它们在离子源和探测器之间的飞行时间来确定5,32。
与其他聚合物表征技术相比, MALDI 的 MS 光谱质量严重依赖于数据采集参数和样品制备。虽然没有设定的样品准备公式, 了解样品准备的每个组分的功能允许更快速的经验优化。MALDI 样品制备中最重要的因素是基体的选择, 因为基体与高分子分析物的相容性对于允许激发矩阵在电离态5中产生单一的吸大分子是至关重要的, 15,17,19。一旦选择合适的基质和 cationization 剂, 就必须确定分析物、基质和 cationization 剂的正确配比。这可以通过在 MALDI 的 MS 目标板 (图 2) 上创建一个二维的样本网格 (图1) 来实现, 并增加一个轴上的矩阵浓度, 并增加 cationization 剂浓度在其他。
与 MALDI 样品制备相似, 没有确定数据采集参数的公式;然而, 应该考虑某些趋势来加速光谱优化。Reflectron 模式, 增加分辨率, 但减少总体信号, 通常是选择较低的质量范围 (在这些例子, 低于 4000 Da), 其中同位素分辨率可以实现。在这些情况下, 采用了 monoisotopic 质量计算和峰值提取方法。对于质量在 4000 Da 以上的高分子样品, 采用线性模式进行平均质量计算和峰值采摘方法。为了提高信号分辨率, 应以较小的增量调整离子源电压, 使大质量聚合物具有较大的电压差 (IS1 与 IS2) 的一般趋势。
虽然优化的样品制备和采集参数可以提供精度, 但质量精度只能通过有效的校准来实现。给定质量的飞行时间在可变采集参数和均匀板位置方面可能会有细微的变化, 因此, 应对每组优化的采集参数进行校准, 以产生准确的质量决心5,30。一旦采集参数和样品准备被优化, 光谱应该使用这些完全相同的条件进行校准。
由于在聚合物的最佳 MALDI 质量谱中观测到的异常分辨率和质量精度, 该技术已成为确定高分子质量分布数据的宝贵的辅助工具。然而, 它在聚合物质量分布范围内解决单个重复单元的能力, 提供了相对于其他聚合物表征技术如凝胶渗透色谱 (GPC) 和核的最终基团分析的特殊优势。磁共振 (核磁共振)。这对于确定端组功能化反应的保真度和端组动词反应的定量性质特别有价值。这篇手稿证明了能够解决的质量的个别聚合物重复单位多达两个小数点的质量的准确性, 使确认的最终小组修改与高度的信心。随着近年来在精密高分子合成领域取得了长足的进步, MALDI 已经成为确定大分子结构和功能的一个越来越重要的工具。

随着生活聚合技术的改进, 具有定量功能的末端基团的精密高分子越来越多地可用1。叠氮化物-炔烃和 thiolene 的并行开发单击化学物质使大分子与其他基团的几乎定量耦合, 提供了多种混合材料2、3、4 的通路..然而, 需要精确的分析技术来表征这些聚合物共轭反应的起始材料和产物。矩阵辅助激光解吸/电离时间的飞行质谱 (MALDI) 是一种宝贵的软电离分析技术的表征聚合物, 因为它可以产生聚合物离子在单一的电荷状态, 最小分裂5,6。MALDI 的 MS 比其他传统的聚合物表征方法有较大的优势, 因为它可以提供质量光谱的分辨率, 在聚合物质量分布的个别 n。因此, 这种质谱可以提供关于平均分子量、重复单位质量和分子量分散性7的精确信息, 这反过来可以阐明相互竞争的聚合机制, 如链转移8.然而, MALDI 的 MS 在提供关于聚合物末端小组9,10的信息是特别强有力的, 可用于证实末端小组修改10,11和其他转换12例如聚合物 cyclizations11,13。同样重要的是, 对质谱分析所需的相对少量分析物 (亚微克) 使得这种技术在仅有微量的材料可用的情况下可以用于表征。
聚合物的 MALDI 分析可以分为四个不同的步骤: 样品制备、仪器标定、光谱采集和数据分析。样品制备是生成最佳 MALDI 质量谱最基本的步骤, 在样品引入到仪器14、15之前发生。选择一个合适的矩阵, 具有相似的溶解度参数作为聚合物分析物是至关重要的, 以获得高质量的 MALDI 质量谱和矩阵选择的指导方针已报告在别处14,15, 16,17。18, 还在网上发表了一份聚合物 MALDI "食谱" 的数据库。对于新型聚合物, 通过首先了解聚合物的溶解度, 选择具有相似溶解度参数14、19的基体, 可以接近基体的选择。具有高质子亲和性的聚合物可以由大多数矩阵14 (通常含有羧酸基团) 质子, 但对于其他聚合物, cationization 剂是必需的14。碱离子与含氧的种类 (例如聚酯和聚醚) 合起来很好, 而不饱和烃 (如聚苯乙烯) 与过渡金属 (如银和铜离子) 加成14,19. 由于本实验中的高分子样品中含有氧原子的骨干, 以钠或三氟乙酸钾 (TFA) 为 cationization 剂。一旦选择了基质和 cationization 剂, 就必须仔细优化分析物、阳离子剂和基体的相对比例, 以确保高信号的噪声。在这个过程中, 样品准备的参数已经被优化, 然而一个经验抽样优化过程 (步骤 1.4.1,图 1), 系统地改变三组分的浓度 (分析物,矩阵和阳离子) 是有效的快速确定其最佳比率。
数据采集还要求对多个参数进行优化。其中最重要的参数包括光谱仪的正负离子模式、仪器操作模式 (线性与反射器)、加速度电压和提取延时时间。另一种可以增加分辨率的方法是利用 "reflectron" 模式20、21、22、23。Reflectron 模式实质上是将离子的飞行路径通过反射飞管末端的离子返回到源附近的探测器上, 同时将离子重新聚焦在不同的动量上, 从而提高分辨率, 尽管降低信号强度。此外, 通过降低激光功率, 减少了碰撞次数和能量, 减少了不均匀性24的破碎和动能, 从而减小了信噪比, 从而可以获得更高的分辨率谱。通过调整所有这些参数, 离子可以集中到最小化的影响, 任何不均匀性的初始位置或速度发生在激光解吸过程。当采集参数优化时, 通常可以达到 1万 Da 以上的离子的同位素分辨率, 但这也取决于飞行管的长度和仪器的设计。大多数含有至少一个杂原子的有机化合物容易与碱性阳离子 (如锂、钠和钾) 络合。许多碱金属是 monoisotopes 或有限同位素, 因此不扩大分布。
虽然可以调整仪器参数以优化数据精度, 但只有在适当的校准11的情况下才能实现数据精度。蛋白质和多肽最初被用作 calibrants 由于其单分散性和可用性, 但遭受可变稳定性和杂质的流行率25。更具成本效益和稳定的替代品包括无机簇和多分散聚合物26、27、28、29。不幸的是, 这些替代品的特点是分散质量, 这会使质量分配复杂化, 而且整体上较小的质量, 这使得它们只对低于 1万 Da 的校准有用。为了对付这些问题, 格雷森和艾尔。25开发了基于大分子的聚酯 MS 校准系统, 单分散, 具有广泛的基体和溶剂相容性, 保质期稳定性 (> 8 年), 降低生产成本。根据该系统的优点, 选择了 calibrant 的实验结果。
校准有两种主要类型: 内部和外部30。在外部校准时, 将分析物支架的质量标准放置在 MALDI 靶板上的不同样品位置上, 而非分析物, 以产生一个单独的质量频谱, 可从中生成校准文件。另一方面, 通过内部校准, 通常可以提高精确度, 这包括将 calibrant 与分析物混合, 以获得 calibrant 和分析物信号的混合光谱。在下面描述的过程中, 执行了外部校准。在对质量尺度进行适当的标定后, 可以获得准确的分析物质量数据。为了保证最精确的标定, 在校准后不久就会发生数据采集。
最后, 得到了优化后的标定数据集, 并对数据进行了分析, 提供了聚合物样品的结构信息。在聚合物分布范围内的 n 场之间的间隔可以提供精确的重复单位质量的测量。数字平均分子量 (mn) 和其他质量分布计算 (例如, 重量平均分子量) 和Đ (分散性)) 也可以确定从信号分布的质量谱 (步骤4.2 计算)。也许最独特的, 在均聚物的情况下, 可以通过确定聚合物分布对重复单位质量的偏移量来确认最终基团质量的总和。信息丰富的 MALDI 的质量谱提供了宝贵的特征数据, 补充了更多传统的聚合物表征技术, 如尺寸排除层析, 傅里叶变换红外光谱, 和核磁共振。

<table><tbody><tr><td>polyoxyethylene bis(azide) (Mn=2000)</td><td>MilliporeSigma (Aldrich)</td><td>689696</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/689696?lang=en&amp;amp;region=US</td></tr><tr><td>poly(ethylene glycol) 2-amino-ethyl ether acetic acid (Mn= 5000)</td><td>MilliporeSigma (Aldrich)</td><td>757918</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/757918?lang=en&amp;amp;region=US</td></tr><tr><td>poly(L-lactide), thiol terminated (Mn=2500)</td><td>MilliporeSigma (Aldrich)</td><td>747386</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/747386?lang=en&amp;amp;region=US</td></tr><tr><td>SpheriCal&amp;reg;&amp;nbsp; peptide low</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>PFS20</td><td>http://www.sigmaaldrich.com/catalog/product/sial/pfs20?lang=en&amp;amp;region=US</td></tr><tr><td>SpheriCal&amp;reg;&amp;nbsp; peptide medium</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>PFS21</td><td>http://www.sigmaaldrich.com/catalog/product/sial/pfs21?lang=en&amp;amp;region=US</td></tr><tr><td>SpheriCal&amp;reg;&amp;nbsp; peptide high</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>PFS22</td><td>http://www.sigmaaldrich.com/catalog/product/sial/pfs22?lang=en&amp;amp;region=US</td></tr><tr><td>2,4 dinitrofluorobenzene</td><td>TCI</td><td>A5512</td><td></td></tr><tr><td>maleimide</td><td>MilliporeSigma (Aldrich)</td><td>129585</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/129585?lang=en&amp;amp;region=US</td></tr><tr><td>1-ethynylfluorobenzene&amp;nbsp;</td><td>Fisher Scientific</td><td>766-98-3</td><td></td></tr><tr><td>triethylamine</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>471283</td><td>http://www.sigmaaldrich.com/catalog/product/sial/471283?lang=en&amp;amp;region=US</td></tr><tr><td>N,N,N&#039;,N&quot;,N&quot;-pentamethyldiethylenetriamine</td><td>MilliporeSigma (Aldrich)</td><td>369497</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/369497?lang=en&amp;amp;region=US</td></tr><tr><td>Copper(I)Bromide</td><td>MilliporeSigma (Aldrich)</td><td>254185</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/254185?lang=en&amp;amp;region=US</td></tr><tr><td>glacial acetic acid</td><td>Fisher Scientific</td><td>A38212</td><td></td></tr><tr><td>sodium metabisulfite</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>13459</td><td>http://www.sigmaaldrich.com/catalog/product/sigald/13459?lang=en&amp;amp;region=US</td></tr><tr><td>potassium trifluoroacetate</td><td>MilliporeSigma (Aldrich)</td><td>281883</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/281883?lang=en&amp;amp;region=US</td></tr><tr><td>trans-2-[3-(tert-butylphenyl)-2-methyl-2-properylidene]malononitrile</td><td>MilliporeSigma (Aldrich)</td><td>727881</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/727881?lang=en&amp;amp;region=US</td></tr><tr><td>a-cyano-4-hydroxycinnamic acid</td><td>MilliporeSigma (Sigma)</td><td>C8982</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/c8982?lang=en&amp;amp;region=US</td></tr><tr><td>tetrahydrofuran</td><td>Fisher Scientific</td><td>T425-1</td><td></td></tr><tr><td>dichloromethane</td><td>VWR Analytical</td><td>BDH1113-4LG</td><td></td></tr></tbody></table>

characterization of synthetic polymers via matrix assisted laser desorption ionization time of flight (maldi-tof) mass spectrometry

在合成均聚物的表征中, 有许多技术可供使用, 但很少有人能为最终群分析提供有用的信息, 因为它是飞行质谱 (MALDI MS) 的基质辅助激光解吸电离时间。本教程演示了使用 MALDI 的合成聚合物的样品制备、光谱采集和数据分析的优化方法. 样品制备过程中的关键参数包括基体的选择、确定适当的 cationization 盐, 并调整基质、阳离子和分析物的相对比例。采集参数, 如模式 (线性或反射器), 极化 (正或负), 加速度电压和延迟时间, 也很重要。给出了合成聚合物的一些化学知识, 同时优化了数据采集参数和样品制备条件, 获得了足够的分辨率和质量精度, 使其具有明确的除重复单位质量和整体分子量分布外, 大多数均聚物 (质量低于 1万) 的最终组的测定。虽然在一组有限的聚合物上显示, 这些一般技术适用于更广泛的合成聚合物, 以确定质量分布, 但最终群体的决心是可能的均聚物与狭窄的分散性。

样品 1: 用三氟乙酸钾作为 HCCA 基质的 cationization 剂, 分析了聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000) (图 3) 的样品。光谱显示了预期的 K+加合物以及从残留 Na+观察到的。
MALDI 的 MS 证实了聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000) 的窄分布 (图 3)。由于 monoisotopic 峰 (完全包括最丰富的元素同位素, 即12C、 1H 16O 和14N) 没有充分解决, 以使其识别, 使用采摘峰值协议,确定每个峰的整个同位素分布的平均质量。同样, 所有的理论计算都是用平均的, 而不是 monoisotopic 的, 每个元素的质量来确定的。利用步骤4中的方程, 用分析软件计算聚合物质量分布的以下特征: mn: 4700, mw: 4710, Đ: 1.00。
为了确认最终组的身份, 选择了一个单独的 n (104) 进行进一步分析 (图 4)。与质量分布计算一样, 由于 monoisotopic 峰值无法解析, 因此在随后的计算中使用了平均质量值。聚乙二醇 2-氨基乙基醚乙酸104的理论质量值由重复单元的质量组成 (44.0530 x 104) 加上α胺端组 (+ 16.02300) 的质量和ω-羧基端组 (升 59.0440) 的质量加上钾阳离子的质量 (升 39.09775), 其产生总 104-4695.67675 的质量。考虑到平均质量计算的精度, 104 的观测质量值为 4695.5, 符合理论值。频谱中较小的偏移峰值与钠的聚合物电离相对应, 其中104的理论质量值由重复单元的质量 (44.0530 x 104) 加上α胺端组 (+ 16.02300) 的质量加上ω-羧基端组 (+ 59.0440) 的质量加上钠阳离子 (+ 22.98922) 的质量, 给出了总104的质量4679.56822。观测到的质量值为104的 + Na+是 4679.4, 只有 0.2 Da 不同于理论价值。可以通过测量多个峰值的平均值来确定更准确的端群质量, 并在别处讨论了11。
聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000) 样品保持其狭窄的分布时, 有选择地功能化反应 (图 5) 与 24 dinitrofluorobenzene (DNFB) (图 6)。光谱表现为加合物钠, 并以 HCCA 为基质。
MALDI 在用 DNFB 修饰时, 证实了聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000) 的窄分布 (图 6)。利用步骤4中的方程, 用分析软件计算聚合物质量分布的以下特征: mn: 4940, mw: 4950 Đ: 1.00。
为了确定聚 (乙二醇) 2-氨基乙基醚乙酸 (Mn = 5000) 的完全功能化是否发生了与 DNFB, 分配的一个单独的 n 的分布被选择了为分析 (图 7)。聚乙二醇 2-氨基乙基醚乙酸功能性104的理论质量与 24-dinitrofluorobenzene 的反应是由 44.0530 x 104 (重复单位的质量) + 182.115 (α胺群的质量反应与 24-dinitrofluorobenzene) + 59.044 (羧基组的质量) + 22.98922 (钠阳离子的质量) = 4845.66022。n = 104 的观测质量值为 4845.8, 与理论值有-0.1 大差异。这种在理论和观察值之间的密切协议表明了对产品的起始材料的完全修改, 但更重要的是, 缺少与起始材料相关的信号, 4811.72722 和 4855.78022, 这质量范围, 或任何额外的副产品证实胺的定量选择性功能化。第二个峰值是观测到的 4823.8, 它匹配的功能性聚合物的 103, 但随着质子在羧酸端组的损失, 与另一个钠离子配合, 与另一种理论质量的 4823.58899, 有差异-0.2 Da。
样品 2: 以三氟乙酸钠为 cationization 剂, 以 HCCA 为基质, 并仅显示预期的钠+加合物, 对聚氧乙烯二 (叠氮化物) (Mn = 2000) (图 8) 进行了样品分析。
由于在这个较低的质量范围内实现了这一分辨率, 因此可以很容易地解决每个 n 个市场的 monoisotopic 峰值, 因此选择了一个 monoisotopic 峰值拾取协议 (平均仅在同位素分布中的第一峰值的质量信号) 和所有相应的计算都利用了每个元素的 monoisotopic 质量。MALDI 证实了聚氧乙烯二 (叠氮化物) 的窄分布 (图 8) (Mn = 2000)。利用步骤4中的方程, 用分析软件计算聚合物质量分布的以下特征: mn: 1940, mw: 1950, Đ: 1.01。
为了确认最终组功能化, 选择了一个单独的 n (42) (图 9)。与以上所确定的质量分布一样, 由于 monoisotopic 峰在每一个 monoisotopic 的同位素分布中都得到了很好的解决, 所以使用了大量的物质。聚氧乙烯二 (叠氮化物) 42 的理论质量值对应于 44.02621 x 42 (重复单位的质量) + 42.00922 (叠氮基端组的质量) + 70.04052 (azidoethyl 端组的质量) + 22.98922 (钠阳离子的质量) = 1984.13978。n = 42 的观测质量值为 1983.95, 与理论值有0.19 大差异。应该指出的是, 特别是在较高的激光功率下, 叠氮化物的功能可以呈现亚稳态片段;然而, 这在具体情况31没有被观察到。
用铜催化叠氮化物-炔烃 cycloaddition 反应 (图 10) 与 1-乙炔-4 fluorobenzene(EFB)进行选择性功能化时, 聚氧乙烯双 (叠氮化物) (Mn = 2000) 样品保持了其狭窄的分布。(图 11) 产生一个 4 fluorophenyltriazolyl (FPT) 组。以三氟乙酸钠为 cationization 剂, 以 HCCA 为基质, 该光谱表现出预期的 Na+加合物。
MALDI 在 EFB 后证实了聚氧乙烯二 (叠氮化物) (Mn = 2000) 的窄分布(图 11) 。利用步骤4中的方程, 用分析软件计算了以下聚合物特征: mn: 2240, mw: 2250, Đ: 1.00。
为了确认样品的完全功能化, monoisotopic 质量被用来分析所选的个体 (42) (图 12)。聚氧乙烯二 (叠氮化物) 42 的理论质量值与 1-乙炔-4-fluorobenzene 对应于 44.02621 x 42 (重复单位的质量) + 162.04675 (FPT 端组的质量) + 190.07805 ( FPT 乙基端组的质量与 1-乙炔-4-fluorobenzene) + 22.98922 (钠阳离子的大量) = 2224.21484。n = 42 的观测质量值为 2224.16, 与理论值有0.05 大差异。
样品 3: 以三氟乙酸钠为 cationization 剂, 对聚乳酸 (l-丙交酯)、硫醇终止 (Mn = 2500) (图 13) 进行了样品分析,仅以加合物和 DHB 为基质。
MALDI 的 MS 证实了聚乳酸 (l-丙交酯) 的狭窄分布, 硫醇终止 (Mn = 2500) (图 13).利用步骤4中的方程, 用程序分析计算了以下聚合物特征: mn: 2310, mw: 2360, Đ: 1.02。
为了确认样品的完全功能化, monoisotopic 质量被用来分析所选的个体 (26) (图 14)。聚 (l-丙交酯) 26 的理论质量值, 硫醇终止 (Mn = 2500) 对应于 72.02113 x 26 (重覆单位的大量) + 17.00274 (羟基的大量) + 61.0112 (数量ω-硫醇末端小组) + 22.98922 (钠的大量阳离子) = 1973.55254。n = 26 的观测质量值为 1973.62, 与理论值有-0.07 大差异。一个更小的信号被观察在2045.74 对应于 72.02113 x 27 (重覆的单位的质量) + 17.00274 (羟基末端小组的大量) + 61.0112 (数量ω-硫醇末端小组) + 22.98922 (钠阳离子的大量)。理论质量为 2045.57367, 与观测到的质量相差0.17。这种小强度, 奇数编号重复单位表明酯交换在环开放聚合的乳酸。第三个非常小的峰值在2057.73 被观察到。这是-0.14 Da 不同于聚 (l-丙交酯) 的理论质量与羧酸末端小组 (而不是硫醇末端小组) 与理论质量 72.02113 x 27 (重覆的单位的质量) + 17.00274 (羟基末端小组的质量) + 73.02895 (大量羧酸) + 22.98922 (钠阳离子的质量) = 2057.59142。这种额外的小杂质很可能是在丙交酯单体的开环聚合过程中由水引发的结果。
聚合 (l-丙交酯), 硫醇终止 (Mn = 2500) 样品保持其狭窄的分布时, 有选择地功能化的硫醇烯反应 (图 15) 与 maleimide (图 16)。以三氟乙酸钠为 cationization 剂, 以 DHB 为基质, 该光谱表现出预期的 Na+加合物。
MALDI 的 MS 证实了聚丙交酯的狭窄分布, 硫醇终止 (Mn = 2500) 后与 maleimide 的硫醇烯反应 (图 16).利用步骤4中的方程, 用分析软件计算了以下聚合物特征: mn: 2310, mw: 2340, Đ: 1.01。应注意的是, 与起始材料相比, mn和 mw的减少是由于电离偏倚 (MALDI 的缺点之一)。当对起始材料的修改相对较小 (97 Da 在这个特殊的修改) 和分散性减少后修改后, MALDI 毫秒计算的平均分子量可能会变得不那么准确。
为了确认聚合 (l-丙交酯) 的完全功能化, 硫醇终止 (Mn = 2500) 与 maleimide 通过硫醇烯反应, monoisotopic 质量被用来分析选定的个体 (26) (图 17)。聚 (l-丙交酯) 硫醇26的理论质量值对应于 72.02113 x 26 (重复单位的质量). + 17.00274 (羟基端组的质量) + 158.02757 (与 maleimide 相连的ω-硫醇末端基团的质量) + 22.98922 (质量钠阳离子) = 2070.56891。n = 26 的观测质量值为 2070.54, 与理论值有0.03 大差异。同样的种类电离与钾也被观察在 2086.49, 对应于0.05 大区别形成理论质量。一个非常小的峰值被观察在2167.58 对应于 72.02113 x 28 (重覆单位的质量) + 17.00274 (羟基末端小组的大量) + 72.02168 (羧酸阴离子的大量) + 22.98922 (钠阳离子的大量) + 38.96371 (钾阳离子的大量)。理论质量是 2167.56844, 这是一个-0.01 不同的观测质量, 并表明相同的微量杂质从水的启动, 在开始的材料观察。这种聚合物的电离与一个当量的钠, 钾之一, 并失去了质子。羧酸质子的损耗和两个阳离子的络合是一元羧酸酸功能高分子的一种常见的电离模式。重要的是要注意的是, 对硫醇烯反应产物观察到的质量相同的变化不会发生在这种羧酸终止化合物上, 这进一步表明它缺乏硫醇末端基团进行功能化反应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57174/57174fig1.jpg"> 图 1: 3 x 3 网格用于取样比率测定.使用 3 x 3 格样本, cationization 剂-分析物基质的相对浓度可以有系统地变化, 以经验主义地确定最佳的样品准备。这通常是通过持有一个三变量常量 (15 µL 分析解决方案), 同时增加其他两个 (cationization 代理 (y 轴) 和矩阵 (x 轴)) 组件的数量由一个集合倍数 (3 倍的例子描述)。<a href="/files/ftp_upload/57174/57174fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57174/57174fig2.jpg"> 图 2: MALDI MS 靶板.MALDI 的 ms 靶板是一个金属板, 它将 MALD 的 ms 样本保存在各个井中进行分析。<a href="/files/ftp_upload/57174/57174fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57174/57174fig3.jpg"> 图 3: MALDI 1 样品的质量谱.这一完整的光谱显示了聚乙二醇 2-氨基乙基醚乙酸 (Mn= 5000) 的总分布与 Na+和 K+电离。<a href="/files/ftp_upload/57174/57174fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57174/57174fig4.jpg"> 图 4: 样本1的单个重复单元的 MALDI 质量谱.该谱显示了聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000) 的单个重复单元, 用于端部基团分析。<a href="/files/ftp_upload/57174/57174fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57174/57174fig5.jpg"> 图 5: 1 样品修改反应方案.为确定起始材料的端组, 聚乙二醇 2-氨基乙基醚乙酸与 24-dinitrofluorobenzene (也称为桑格试剂) 反应。<a href="/files/ftp_upload/57174/57174fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57174/57174fig6.jpg"> 图 6: MALDI 1 型改性样品的质量谱.这种全谱显示了聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000) 的整体分布, 具有 24-dinitrofluorobenzene。<a href="/files/ftp_upload/57174/57174fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57174/57174fig7.jpg"> 图 7: MALDI 样本1modification 的单个重复单元的质量谱.为了确认端组功能化, 该谱显示了聚乙二醇 2-氨基乙基醚乙酸 (Mn = 5000) 在 24-dinitrofluorobenzene 反应后的个体重复单位。<a href="/files/ftp_upload/57174/57174fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57174/57174fig8.jpg"> 图 8: MALDI 2 样品的质量谱.这一完整的光谱显示了聚氧乙烯二 (叠氮化物) (Mn = 2000) 电离与 Na+加合物的总分布。<a href="/files/ftp_upload/57174/57174fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57174/57174fig9.jpg"> 图 9: 样本2的单个重复单元的 MALDI 质量谱.该频谱显示了聚氧乙烯双叠氮化物 (Mn = 2000) 的重复单元, 以确认最终组<a href="/files/ftp_upload/57174/57174fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/57174/57174fig10.jpg"> 图 10: 2 样品修改反应方案.为确定起始材料的端组, 聚氧乙烯双叠氮化物 (Mn = 2000) 通过铜催化叠氮化物-炔烃 cycloaddition (CuAAC) 与 1-乙炔-4-fluorobenzene 反应。<a href="/files/ftp_upload/57174/57174fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/57174/57174fig11.jpg"> 图 11: MALDI 2 型改性样品的质量谱.这一完整的光谱显示了聚氧乙烯二 (叠氮化物) (Mn = 2000) 功能化与 1-乙炔 4-fluorobenzene 的整体分布。<a href="/files/ftp_upload/57174/57174fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/57174/57174fig12.jpg"> 图 12: 单个重复单元的 MALDI 的质量谱2例修改.该谱显示了聚氧乙烯双 (叠氮化物) (Mn = 2000) 的单个重复单元与 1-乙炔-4-fluorobenzene 通过铜催化叠氮化物-炔烃 cycloaddition 的反应, 以确认端组功能化。<a href="/files/ftp_upload/57174/57174fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/57174/57174fig13.jpg"> 图 13: MALDI 3 样品的质量谱.全谱显示聚丙交酯的总分布, 硫醇终止 (Mn = 2500)。<a href="/files/ftp_upload/57174/57174fig13large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/57174/57174fig14.jpg"> 图 14: 样本3的单个重复单元的 MALDI 质量谱.该频谱显示了聚 (l-丙交酯) 的个体重复单位, 硫醇终止 (Mn = 2500), 以确认最终组。<a href="/files/ftp_upload/57174/57174fig14large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/57174/57174fig15.jpg"> 图 15: 3 样品修改反应方案.为了确认起始材料的端组, 聚 (l-丙交酯), 硫醇终止 (Mn = 2500) 通过硫醇烯偶联 maleimide 反应。<a href="/files/ftp_upload/57174/57174fig15large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/57174/57174fig16.jpg"> 图 16: MALDI 3 型改性样品的质量谱.这一全谱显示了聚合 (l-丙交酯) 反应产物的总分布, 硫醇终止 (Mn = 2500) 和 maleimide。<a href="/files/ftp_upload/57174/57174fig16large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/57174/57174fig17.jpg"> 图 17: 单个重复单元的 MALDI 的质量谱3例修改.为了确认端组功能化, 该谱显示了聚乳酸 (l-丙交酯) 的个体重复单位, 硫醇在硫醇-烯与 maleimide 反应后终止 (Mn = 2500)。<a href="/files/ftp_upload/57174/57174fig17large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

本文介绍了用矩阵辅助激光解吸电离时间的飞行质谱 (MALDI) 表征合成聚合物的方法, 包括样品制备、光谱采集和数据分析的优化。

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

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Organic Chemistry

Chemistry

Unit 1

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

Synthetic Polymers

SCIENTISTS IN ACTION

25.2K 次观看.  Tulane University. 本文介绍了用矩阵辅助激光解吸电离时间的飞行质谱 (MALDI) 表征合成聚合物的方法, 包括样品制备、光谱采集和数据分析的优化。

视频: Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

河豚有毒改进的聚合酶链反应 - 限制性片段长度多态性分型液相色谱/质谱法

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

科研

遗传学

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

基质辅助飞行的完整蛋白质（MALDI-TOF）质谱分析激光解吸/电离飞行时间大于100 kDa的

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

化学

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

生物工程

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

寡 peptoids 的合成及质谱分析

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

使用循环离子淌度光谱仪进行串联离子淌度实验

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

生物学

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.

Mass Spectrometer: Principle, Operation, Applications

质谱分析法导论

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the ...

教育

JoVE Science Education

分析化学

Matrix-Assisted Laser Desorption Ionization (MALDI)

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and characterization of various biomolecules, including proteins, peptides, nucleic acids, and carbohydrates. MALDI spectrometry is widely employed in biological and medical research, as well as in fields like pharmacology and biochemistry.
The analyte of interest, a biomolecule or a mixture of biomolecules, is mixed with a suitable matrix material. The matrix is usually an organic compound, such as a small organic acid or a crystalline substance, that facilitates ionization. This analyte-matrix mixture is then deposited onto a target surface, inserted into the mass spectrometer, and dried. During drying, the matrix forms small crystals that incorporate the analyte molecules within their structure.
Once the sample is prepared, a pulsed laser is directed onto the target surface. The matrix molecules absorb the laser beam energy, causing them to heat up and vaporize rapidly. This process is known as laser desorption. As the matrix molecules vaporize, they carry the embedded analyte molecules. The vaporized analyte molecules become ionized through a process called proton transfer. The proton transfer occurs between the matrix and the analyte molecule. The matrix molecule donates a proton to the analyte molecule, resulting in the formation of protonated ions. The analyte ions are then accelerated by an electric field and further separated based on their mass-to-charge ratio in a time-of-flight mass analyzer.
The mass spectrum obtained from MALDI spectrometry provides valuable information about the mass and relative abundance of the analyzed biomolecules.

基质辅助激光解吸电离（MALDI）

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and ...

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

摘要

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