这项工作得到了国家科学中心（NCN）在2018/31/D/ST5/00399和OPUS10 2015/19/B/ST7/02163项目SONATA14的部分支持。

1.缺陷选择性蚀刻
<ol><li>固体蚀刻剂制备<ol><li>通过将组成碱氢氧化物和金属氧化物在蒸馏水中溶解和混合，制备氢氧化钾 （KOH） 和氢氧化钠 （NaOH） 强碱与氧化镁 （MgO） 的共晶混合物。分别在 NaOH、KOH 和 MgO 的百分比下保持 53.6/37.3/9.1 的化学计量量13.MgO的添加增加了蚀刻剂的粘度，使其保持在Ga极性表面上，并且不会流过边缘流向N极性表面13,14。所有使用的化学品都应具有实用级质量。</li>
		<li>将热板上的烧瓶中的混合物加热至200°C，并通过磁力搅拌搅拌1小时（高于KOH-NaOH共晶点的熔点）。</li>
		<li>通过降低热板的温度将混合物冷却至~100°C，以完全蒸发剩余的液体。此步骤取决于烧瓶大小和水量，因此可能需要几分钟长达 1 小时。</li>
		<li>将固体蚀刻剂（表示为 E+M）转移到干燥的瓶子中，避免暴露在湿气中。 注意:KOH 和 NaOH 可能会引起皮肤刺激和眼睛损伤。戴上手套和护目镜。该协议也可以在此处暂停。</li>
	</ol></li>
	<li>缺陷选择性蚀刻<ol><li>准备干净的氮化镓表面进行分析。在蓝宝石上外延生长的 GaN 用于以下步骤12.</li>
		<li>将GaN样品放在加热至~450°C的热板上。 将热电偶放在样品附近，以精确读取真实温度。</li>
		<li>将一块固体 E+M 蚀刻剂放在 GaN 顶部并静置 3 分钟。</li>
		<li>从热板中取出样品，放入装有热HCl的烧杯中3-5分钟，以消除剩余的E + M。</li>
		<li>从HCl中取出样品，放入装有去离子（DI）水和超声波浴的烧杯中5-10分钟。</li>
		<li>用 N2 吹干样品。 注意:盐酸可能会引起皮肤刺激和眼睛损伤。戴上手套和护目镜。避免被烫伤。该协议也可以在此处暂停。</li>
	</ol></li>
</ol>2. 扫描电子显微镜（SEM）观察
<ol><li>在样品上做标记（例如，使用金刚石裁刀用L形划痕）。</li>
	<li>使用导电粘合剂（即双面碳导电胶带或类似材料）将样品安装在专用于要使用的 SEM 模型的金属短截面上。在样品制备和转移过程中戴上手套，以避免手上的油脂污染。</li>
	<li>添加步骤 2.2 中的一块胶带，将样品表面与金属短截线连接起来，以防止电荷在样品表面积聚。或者，可以应用带有导电材料（~10 nm 厚）的溅射涂层以防止充电效应。</li>
	<li>获取至少三张高分辨率 SEM 显微照片（理想情况下，至少五张）样品的俯视图。每张图像应显示至少 25 x 25 μm 的面积。避免从具有宏观表面缺陷的表面区域拍摄图像。 图 1 给出了一个典型的结果。</li>
	<li>请注意每张图片相对于 L 形标记的确切位置。 注意:协议可以在此处暂停。</li>
</ol>3. 二次离子质谱测量
<ol><li>工具校准<ol><li>使用负极性、Cs 初级离子以 7-13 keV 冲击能量校准 SIMS 设备。对齐次梁和主梁。保持光束尽可能小（直径至少 1 μm），因为横向分辨率由光束的大小预先确定。</li>
		<li>为具有不同离子电流密度的光束准备五到七个设置。为简单起见，请保持光束的大小不变，并更改光束电流。测量光束电流和光束15 的大小。在以下步骤中，使用5 nA、10 nA、15 nA、20 nA、30 nA和50 nA的束流，光斑尺寸为1 μm。</li>
		<li>使用 50 x 50 μm 光栅大小和 35 x 35 μm 分析区域执行以下步骤。选择 256 x 256 像素以获得空间分辨率。如果未另行指定，则对每个信号使用标准积分时间（通常为 1-2 秒）。</li>
	</ol></li>
	<li>主要来源稳定性<ol><li>选择具有中等束流 （15-20 nA） 的设置。</li>
		<li>使用 30Si2- 二次离子获得空白硅晶圆的一系列图像。对于每个图像，将信号积分 5-10 分钟。</li>
		<li>将所有图像与第一张图像进行像素间比较。如果 >5% 的像素与第一张图像相差 >5%，则表明光束变得不稳定。注意光束稳定性的时间跨度。</li>
	</ol></li>
	<li>测量 注意: 以下步骤是在光束的稳定时间跨度内执行的。<ol><li>按照 Peres 等人描述的程序估计测量室7 中氧气污染的背景水平。对于每次测量，无需获得氧浓度的绝对值，因为 16个 O- 和 69个 Ga- 信号的强度比就足够了。</li>
		<li>使用在步骤 3.1.2 中准备的光束设置。对每个光束设置至少执行五次测量。使用16 O-二次离子获得深度分布，达到~200 nm深度，并通过对信号进行10-15 s的积分来测量69Ga-二次离子的强度。不要在已获得 SEM 图像的区域执行此操作。</li>
		<li>绘制 16 个 O 和 69个 Ga- 信号的强度比作为反转初级电流密度的函数（无需计算绝对值）。预期有良好的线性拟合（此处，R2 = 0.997）。估计真空背景贡献，如 图 2 所示。</li>
		<li>为以下步骤选择强光束 （30 nA）。获取将用于平场校正的图像。将 30Si2- 二次离子用于空白硅晶圆。对信号进行积分 5-10 分钟。 图 3 给出了一个典型的结果。</li>
		<li>在获取 SEM 图像的相同区域执行深度剖面测量。使用 16O- 二次离子，对每个数据点的信号进行 3-5 秒的积分。 注意:协议可以在此处暂停。</li>
	</ol></li>
</ol>4. 数据处理
<ol><li>从深度剖面重建 3D 图像。</li>
	<li>执行平场校正:使用在步骤3.3.4中获得的参考图像对每个 16O- 离子图像进行像素到像素归一化。图 4A 为原始数据， 图4B 为平场校正后的图像。</li>
	<li>从步骤3.3.2中获得的图中估计真空背景贡献。无需计算绝对值;但是，请注意可归因于真空背景贡献的总计数的特定百分比。对于这种实验，通常介于 90-95% 之间的值。</li>
	<li>减去真空背景贡献:随机消除 90-95% 的 16 个 O 计数。 图4C 给出了单个平面的典型结果。</li>
	<li>将剩余计数绘制为 3D 图像。 图 5 给出了一个典型的结果。</li>
	<li>使用任何支持图层的图像编辑器软件，集成来自所有数据点的信号，并将 2D 图像叠加在先前获得的 SEM 图像上。将 SEM 图像视为背景。包含 SIMS 结果的图层应仅包含彩色像素的实际计数（删除中间的白色区域）。向此图层添加 ~30% 的透明度。 图 6 显示了一个典型结果</li>
</ol>

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作者没有什么可透露的。

探测器的不均匀性和真空背景贡献问题很容易通过平场校正和寄生计数的减法来解决。减法程序并不完美，因为它可能会减去氧气聚集的贡献。相反，在另一个位置，它将使背景计数不受影响;因此，一些人为计数可能仍然存在，而一些实际计数会减少。然而，它足够高效和灵敏，可以提供可接受的结果。
初级光束不稳定性是最成问题的，因为初级光束参数的劣化会使二次离子图像模糊;因此，无法获得有关样品的可靠信息。协议中的第3.2节尤为重要。例如，对于对准良好的光束，第一个 30Si2- 二次离子图像反映了检测器的不均匀性，但一段时间后，图像将开始发生变化。这是由初级光束参数的劣化（即初级电流损耗、散焦、位置漂移等）引起的。因此，估计光束稳定性的时间跨度非常重要。建议在光束初始化后 2-3 小时开始实验，因为它通常更稳定。
如果实验是在光束的稳定时间跨度内进行的，但结果仍然不令人满意，建议考虑主光束的质量。对于较小的初级光束，仅通过观察二次离子图像来确认足够的质量更具挑战性。因此，建议在溅射~1μm非常平坦的材料（即空白硅晶圆）后，在陨石坑底部进行原子力显微镜粗糙度测试。如果均方根粗糙度大于 1 nm，则需要进一步优化主光束。
光束的大小限制了该方法的横向分辨率。SIMS可以对比束尺寸小的特征进行成像，但二次离子图像将继承一次离子束的形状和大小。如果两个特征之间的距离小于光束的大小，二次离子图像将使它们模糊在一起。尽管存在这些问题，该方法仍允许用户获得固态样品中杂质/掺杂剂的真实 3D 分布。此外，原子的任何空间偏析都可以与缺陷和界面的位置相关联。
对于基于 GaN 的结构（即氧装饰），作为局部非辐射复合中心的位错负责 n 型电导率。对于其他材料，掺杂剂/污染原子分布的任何不均匀性都可能对器件的性能产生重大影响。因此，该协议对于生长和处理程序的故障分析和优化特别有用。

二次离子质谱（SIMS）是一种用于污染监测的知名技术，具有出色的检测限1,2,3,4,5,6。对于轻元素（例如氢、碳、氮、氧）来说，真空背景贡献可能是有问题的，这些元素可能以残余气体的形式存在于测量室中。Peres等人之前建立了一种估计背景贡献的技术;因此，可以确定污染原子的实际浓度7.
在许多材料中，污染原子的分布并不均匀。氮化镓（GaN）的情况特别有趣，因为据预测，氧主要装饰螺杆和混合位错8,9,10,11。考虑到大多数分析方法缺乏检测低浓度污染原子的灵敏度或空间分辨率，因此必须开发一种能够对分离杂质进行 3D 定位的 SIMS 测量程序12。
虽然许多SIMS光谱仪都配备了位置敏感探测器，但深度剖面的直接三维（3D）重建不足以获得氮化镓样品中氧原子的真实分布。探测器的缺陷可能会使图像失真，并阻止研究人员获得污染原子的真实分布。然而，一个很大的问题是真空背景贡献，因为通常>90%的登记氧计数来自分析室中存在的残余气体。这里介绍的是一种识别和充分解决这些挑战的方法。
探测器的不均匀性可以在空白硅晶圆上进行测试。由于微通道板检测器中每个通道的灵敏度不同，即使是较长的积分时间也会导致观察到一些二次离子图像的不均匀性。因此，需要平场校正来获得偏析原子三维分布的高质量图像。
真空背景贡献与从真空吸附到分析区域的污染原子通量有关。考虑到该过程是动态的（即样品表面不断被主光束溅射），可以假设分析区域的每个点吸附这些氧原子的概率相同。此外，它们几乎立即被溅射，没有足够的时间进行分离。因此，统计方法是最有效的。随机消除 90%（或更多）的氧计数应揭示氧聚集的区域。
应该注意的是，主光束的稳定性对于这种类型的实验至关重要。一段时间后，光束的强度和均匀性会变差，从而降低图像质量。因此，必须估计光束稳定运行的时间跨度，并在光束变得不稳定之前进行所有实验。该协议可以很容易地用于其他材料和检测预计分布不均匀的元素。将其与湿化学蚀刻相结合特别有趣，湿化学蚀刻揭示了位错的位置和类型。因此，团聚杂质的位置可以与缺陷的位置相关联。

<table border="0" cellpadding="0" cellspacing="0" style="width:654px;" width="652">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Heating plate with ceramic top plate</td>
			<td style="width:136px;">IKA - Werke GmbH</td>
			<td align="right" style="width:123px;">3644200</td>
			<td style="width:172px;">for defect selective etching; yellow MAG HP 7</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Hydrochloric acid (HCl) solution 35-38%</td>
			<td style="width:136px;">Chempur</td>
			<td align="right" style="width:123px;">115752837</td>
			<td style="width:172px;">for etchant removal; pure p.a.; CAS: 7647-01-0</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:57px;width:223px;">Magnesium oxide (MgO)</td>
			<td style="width:136px;">Chempur</td>
			<td align="right" style="width:123px;">116140200</td>
			<td style="width:172px;">for eutectic solid etchant prepration; pure p.a.; CAS: 1309-48-4</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:57px;width:223px;">Potassium hydroxide (KOH)</td>
			<td style="width:136px;">POCH S.A.</td>
			<td align="right" style="width:123px;">746800113</td>
			<td style="width:172px;">for eutectic solid etchant prepration; pure p.a.; CAS: 1310-58-3</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:57px;width:223px;">Sodium hydroxide (NaOH)</td>
			<td style="width:136px;">POCH S.A.</td>
			<td align="right" style="width:123px;">810925112</td>
			<td style="width:172px;">for eutectic solid etchant prepration; pure p.a.; CAS: 1310-73-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Secondary ion mass spectrometer</td>
			<td style="width:136px;">CAMECA</td>
			<td style="width:123px;">IMS SC Ultra</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scanning electron microscope</td>
			<td style="width:136px;">Hitachi</td>
			<td style="width:123px;">SU8230</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d depth profile reconstruction of segregated impurities using secondary ion mass spectrometry

所提出的方案结合了使用二次离子质谱（SIMS）的出色检测限（1 ppm至1 ppb）和合理的空间分辨率（~1 μm）。此外，它还描述了如何获得固态材料中分离杂质/掺杂剂的真实三维 （3D） 分布。由于与SIMS相关的测量伪影，通常很难实现直接的3D深度剖面重建。这里介绍的是一种识别和解决这一挑战的方法。讨论了三个主要问题，包括 i） 通过平场校正补偿探测器的不均匀性;ii） 估计和减去真空背景贡献（分析室中存在的残余气体中的寄生氧计数）;iii）在一次离子源的稳定时间跨度内执行所有步骤。湿化学蚀刻用于揭示材料中位错的位置和类型，然后将SIMS结果叠加在通过扫描电子显微镜（SEM）获得的图像上。因此，团聚杂质的位置可能与某些缺陷的位置有关。该方法速度快，不需要复杂的样品制备阶段;然而，它需要高质量、稳定的离子源，并且必须快速进行整个测量，以避免主光束参数恶化。

在3D图像中应观察到非常清晰的柱状结构。更多的氧气应该聚集在靠近表面的区域，因为蚀刻过程会引入更多的氧气，这些氧气可以通过样品扩散。 图 7 显示了原始数据的 3D 图像和还原过程如何显示最终结果的动画。 图4C 还给出了单个平面的典型结果。
叠加在SEM图像上的SIMS图像显示，氧气沿着最大蚀刻坑的岩心聚集。这些可归因于混合/螺钉位错15。需要注意的是，如果核心小于主光束的尺寸，则次映像将继承主光束的尺寸和形状。在次优实验中，可以看到氧计数的随机分布（图8）。 图 9 显示了光束在实验过程中变得不稳定的情况。具体来说，对于如此接近地表的区域来说，质量很高，但在实验过程中会逐渐恶化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61065/61065fig1.jpg"> 图 1:使用 E+M 蚀刻在 GaN 表面显示的蚀刻坑的 SEM 显微照片。 将蚀刻参数设置在450 °C下3分钟。插图描绘了一张放大的显微照片，揭示了位错岩心上产生的六边形凹坑。两个最大的凹坑 （>500 nm） 表示具有 Burgers 矢量螺杆分量的位错。此图经许可转载12. <a href="https://www.jove.com/files/ftp_upload/61065/61065fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61065/61065fig2.jpg"> 图2:平均O-浓度与反向初级电流的关系。 真空背景贡献可以从图中估计出来。误差线表示每个数据集的标准偏差（五个测量值）。 <a href="https://www.jove.com/files/ftp_upload/61065/61065fig2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61065/61065fig3.jpg"> 图 3:空白硅晶圆的典型 30Si2- 二次离子图像。 强度差异是由探测器的不均匀性引起的。<a href="https://www.jove.com/files/ftp_upload/61065/61065fig3large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61065/61065fig4v2.jpg"> 图 4:在 3D 模式下测量的氧计数分布的典型平面图。 显示的图像 （A） 来自原始数据，（B） 平场校正后，以及 （C） 减去真空背景贡献后的图像。此图经许可改编12. <a href="https://www.jove.com/files/ftp_upload/61065/61065fig4large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61065/61065fig5.jpg"> 图 5:5 μ m x 5 μ m x 1 μm 长方体中氧气计数的 3D 视图。 为了获得更好的可见性，z 刻度被拉长。有关动画，请参阅补充图 1。此图经许可改编12.<a href="https://www.jove.com/files/ftp_upload/61065/61065fig5large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61065/61065fig6.jpg"> 图6: 投射在SEM显微照片上的氧二次离子（蓝色像素）的横向分布。 尽管存在与SIMS相关的伪影（由主光束的大小决定的横向分辨率），但观察到最大凹坑的位置与氧气之间存在明显的相关性。此图经许可改编12. <a href="https://www.jove.com/files/ftp_upload/61065/61065fig6large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61065/61065fig7.jpg"> 图 7:显示如何执行还原过程的动画。 在程序开始时，所有计数都存在，然后对于每一层，随机消除 90% 的计数。 <a href="https://www.jove.com/files/ftp_upload/61065/61065fig7.mp4" target="_blank">请点击此处下载此动画。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61065/61065fig8.jpg"> 图8:次优实验中氧计数的随机分布。 <a href="https://www.jove.com/files/ftp_upload/61065/61065fig8large.jpg" target="_blank">请点击这里查看此图的较大版本。</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61065/61065fig9.jpg"> 图 9:使用不稳定光束进行的实验。 质量随着溅射深度的增加而下降。<a href="https://www.jove.com/files/ftp_upload/61065/61065fig9large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图 1. <a href="https://www.jove.com/files/ftp_upload/61065/Supplemental_Figure_1.mp4" target="_blank">请点击这里下载此图。</a>

Watch this Scientific Journal Video about 3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry at JoVE.com

3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

所提出的方法描述了如何识别和解决与二次离子质谱相关的测量伪影，以及如何获得固态材料中杂质/掺杂剂的真实 3D 分布。

使用二次离子质谱法重建分离杂质的 3D 深度剖面

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial ...

3d-depth-profile-reconstruction-segregated-impurities-using-secondary

Research

JoVE Journal

Chemistry

1.6K 次观看.  Łukasiewicz Research Network-Institute of Electronic Materials Technology. 所提出的方法描述了如何识别和解决与二次离子质谱相关的测量伪影，以及如何获得固态材料中杂质/掺杂剂的真实 3D 分布。 

视频: 3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

科研

化学

Analyzing the Photo-oxidation of 2-propanol at Indoor Air Level Concentrations Using Field Asymmetric Ion Mobility Spectrometry

We demonstrate a versatile protocol to be used for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) volatile organic carbons (VOCs), illustrating this with a titanium dioxide based catalyst, and the VOC 2-propanol. The protocol takes advantage of field asymmetric ion mobility spectroscopy (FAIMS), an analysis tool that is capable of continuously identifying and monitoring the concentration of VOCs such as 2-propanol and acetone at the ppb level. The continuous nature of FAIMS allows detailed kinetic analysis, and long-term reactions, offering a significant advantage over gas chromatography, a batch process traditionally used in air purification characterization. The use of FAIMS in photocatalytic air purification has only recently been used for the first time, and with the protocol illustrated here, the flexibility in allowing alternative VOCs and photocatalysts to be tested using comparable protocols offers a unique system to elucidate photocatalytic air purification reactions at low concentrations.

A protocol for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) model volatile organic carbons such as 2-propanol is described.

用场不对称离子迁移光谱法分析 2-丙醇在室内空气浓度中的光氧化作用

We demonstrate a versatile protocol to be used for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) volatile ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

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 ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

基质辅助激光解吸/电离质谱中增强碳水化合物离子信号的高效样品制备方法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

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 ...

Imaging Corrosion at the Metal-Paint Interface Using Time-of-Flight Secondary Ion Mass Spectrometry

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), illustrating that SIMS is a suitable technique to study the chemical distribution at a metal-paint interface. The painted Al alloy coupons are immersed in a salt solution or exposed to air only. SIMS provides chemical mapping and 2D molecular imaging of the interface, allowing direct visualization of the morphology of the corrosion products formed at the metal-paint interface and mapping of the chemical after corrosion occurs. The experimental procedure of this method is presented to provide technical details to facilitate similar research and highlight pitfalls that may be encountered during such experiments.

Time-of-flight secondary ion mass spectrometry is applied to demonstrate the chemical mapping and corrosion morphology at the metal-paint interface of an aluminum alloy after being exposed to a salt solution compared with a specimen exposed to air.

利用飞行时二次离子质谱法成像金属颜料界面的腐蚀

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.