我们感谢金融支持从加拿大基因和基因组不列颠哥伦比亚省 (204PRO) 的操作和技术发展 (214PRO) 通过基因组创新网络 (GIN)。我们感谢研究所的麦吉尔大学健康中心拍摄 MALDI TOF 仪用于药物发现平台。H.L.是感谢支持从国家科学和工程研究理事会的加拿大 (NSERC) 博士后。C.H.B 非常感谢支持从前缘养老基金 （叶）。C.H.B.是感谢支持从在麦吉尔大学 （加拿大，魁北克，蒙特利尔） 分子肿瘤学的西格尔麦吉尔椅子。M.X.C.和 C.H.B.都很感激支持沃伦 Y.索珀慈善信托基金和阿尔文 · 西格尔家族基金会往犹太医院 （蒙特利尔，魁北克，加拿大）。

试剂如下所述的数额基于 20 病人血浆样品的测量。议定书 》 提出了以下如下准则的维多利亚大学 &#39; s 人类研究伦理委员会。
 1.昂代我人血浆中 <ol><li>解冻血浆样品 (&#8805; 200 微升) 在一个房间里温度水浴 5 分钟，然后把样品直到完全融化的冰上。</li>
	<li>每个样品的浓度手动来分离 1.1 毫升深孔板 （样板） 井和离心 10 分钟在 2 ° C 和 1278 x g.离心机板转移 200 微升</li>
	<li>使用自动化的液体处理系统连续稀释 500 fmol/微升 Ang I NAT 标准溶液准备与鸡卵清白蛋白的六个校准器解决方案 (0.1、 0.2、 0.6、 1.9、 5.7，17.2 fmol/微升)。</li>
	<li>的每个校验仪对这一代人很好，和 125 µ L 移液管 200 微升缓冲区来样来板。 
	注意: 在磷酸盐缓冲的液 (PBS) 缓冲区需要新鲜准备实验日 CEWA.</li>
	<li>使用自动化的液体处理系统，在一个新的盘拌 125 µ L 的等离子体上清液或 125 µ L 的 PBS CEWA 25 微升的昂我代缓冲区，包含 1 M 三、 0.2 毫米乙二胺四乙酸 (EDTA) 和 1 毫米苯甲基磺酰氟 (PMSF)。 
	注: 准备 Ang 我代缓冲区由搅拌 1 M 三/0.2 毫米 EDTA 缓冲溶液 (适应 pH 5.5 使用醋酸) 和 PMSF 解决方案 （在甲醇中的 100 毫米）。两种解决方案都可以被存储到 1 个月在 4 ° C.混合两个解决方案日实验。</li>
	<li>自动转移到 96 孔板，3 复制每个解决方案，每井 34 微升的解决方案。孵化在 37 ° C 为 3 h.板</li>
</ol> 2。免疫富集的昂我使用抗体包裹小珠 <ol><li>上磁珠 G 蛋白抗体 注: 抗体与珠的共轭手动术 3 h 昂我代期一天的实验。其他分析物，共轭的珠子可能能够存储在 PBS 缓冲含有 0.015%章 (PBSC) 在 4 ° C 为三个月或更长时间，取决于性能和稳定性的抗体。
	<ol><li>珠浆 （足够测量 20 样品和制作 6 点标准曲线） 的转让 110 微升到 1.5 毫升管。洗珠七次 1 毫升的 25%乙腈/外周血干细胞，与三次 1 毫升的外周血干细胞。使用磁性表座颗粒之间每清洗一步珠。在去年华盛顿后移除洗涤缓冲液 
		注: 洗 7 倍 25%乙腈/外周血干细胞是删除不兼容的 MS 添加剂中珠浆，如吐温-20 的关键。如果所选的珠子有没有这种添加剂，可能不需要此广泛洗涤步骤。</li>
		<li>重珠悬在 110 µ L 的外周血干细胞，并添加的抗 Ang 110 µ L 我抗体 (最后抗体浓度: 100 微克/毫升)。吹打，混合的珠子和解决方案，然后孵化它们在室温下 1 h，旋转在 8 rpm。</li>
		<li>洗珠 3 次 1 毫升的外周血干细胞，并在中央政治局常委会的 1100年微升。 
		注: 如果任何珠解决方案孵化期间流入的管帽，降速使用台式离心机在 2680 x g.解决方案</li>
		<li>手动将珠解决方案转移到 96 孔板 （珠标准板）。使用自动化的液体处理系统对分装至同一 96 孔板，每井 120 微升珠</li></ol></li> <li>免疫富集在珠子上 <ol><li>3 h 昂我代期间，放在孵化盘后10 分钟终止代昂 I.冰</li>
		<li>自动稀释 100 倍 SIS 肽原液 (10 皮摩尔/μ L) 用 PBS 缓冲，并进一步自动分装到 96 孔板 PCR 板稳定 isoptope 标准肽稀释。将 1.5 微升的 （含 100 fmol） 的稳定同位素标准 (SIS) 肽溶液转移到孵化板每个井和混合与血浆样品或 PBS 缓冲区中的 CEWA.</li>
		<li>自动将孵化盘的内容转移到珠解决方案，每井 10 毫升，混合。</li>
		<li>以 8 rpm 旋转，同时孵化在 4 ° C 1 h 板。</li>
		<li>珠子三次自动用 5 毫米碳酸氢铵 (AmBic) 溶液冲洗，每次洗涤井每 100 μ L。在最后一次冲洗后, 重 7 微升 5 毫米 AmBic 解决方案在每口井的悬珠。使用一块磁铁把珠拉到底部后最后华盛顿州</li>
	</ol></li></ol> 3。转移到 MALDI 靶板和添加矩阵解法珠 <ol><li>转让 7 微升的珠浆自动到 MALDI 靶板上配有现货 2 600 微米。让珠子干涸。 
	注意: 一个小的 USB 供电风扇可以用来加速干燥过程珠。</li>
	<li>自动添加 2 µ L 的 α-氰基-4-羟基肉桂酸 （本病）-从好到现场对靶板每个样本矩阵的矩阵解法 （包含 3 毫克/毫升本病、 1.8 毫克/毫升柠檬酸铵、 70%乙腈和 0.1%三氟乙酸）.</li>
</ol> 4。信号采集的 MALDI TOF MS 和数据分析 <ol><li>分析样品点与 MALDI TOF 仪器使用积极的反射模式。执行内部校准、 数据平滑和特定供应商的软件自动基线减法。 
	注意: 选择的模式 （正/负线性/反射） MALDI TOF 测量取决于目标多肽或蛋白质。</li>
	<li>计算相对响应比 （Nat/SIS 强度比），并进行比较的标准曲线确定的李安我每个样品中的浓度。计算方程 (1)，在那里的 PRA δ t Ang 我代 表示的时间用于生成 Ang I. 
	PRA = [昂我] / δ t Ang 我代 （1)</li></ol>

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C.H.B 拥有专利的 iMALDI 技术。

相比传统的基于 MS 蛋白质定量，iMALDI 使用抗体来丰富分析物和净化他们从复杂的样品，因此使它能够量化的蛋白质或多肽在低浓度时。IMALDI 协议的关键一步是目标肽免疫富集。为此目的，应选择具有高特异性和亲和力抗体。在 SISCAPA，报道在 10-9米或更好的抗体亲和力将需要实现高灵敏度低吴/毫升21。此外，与高的抗体结合能力的珠子是首选最优浓缩。
<p class="jove_co...

质谱技术已经成为不可或缺的工具，在定量蛋白质组学。质谱分析可以确定目标蛋白质或多肽的群众，因此所得的分析物信号可以高度特异性相比于免疫分析。两种电离方法，电喷雾串联质谱和 MALDI，常被用于检测蛋白质和多肽1，，23，4。在基于 MS 蛋白质定量的主要挑战在于复杂样品在吴/毫升或 pg/mL 的浓度在高丰度蛋白低丰度蛋白的检测及血浆中发现很多候选人蛋白质生物标志物在这范围之内5。这个问题很大程度上造成的人类蛋白质组6复杂性与本质上宽动态范围。
为了克服这些检测，免疫色谱-质谱方法已充实目标蛋白质或多肽从样品溶液在固体的表面，紧接着洗脱物和 MS 测量7，8,9,10.通过免疫浓缩物都从复杂样品纯化，因此从其他分子离子抑制影响降至最低。各种固体支架间磁珠是目前使用最广泛那样高的抗体结合能力的优势和易用性的处理。磁珠与不同的 functionalizations 和大小有被开发和商业化免疫沉淀实验。到目前为止，免疫浓缩制成的小珠已经配套与电喷雾电离 (ESI) 和 MALDI 质谱蛋白质和多肽的测量。稳定同位素标准和捕获由抗多肽抗体 (SISCAPA) 技术，在样品中的蛋白质消化，紧接着孵化与抗体包裹小珠免疫富集。在"古典"SISCAPA，捕获的 proteotypic 多肽是从珠、 洗脱，由液相色谱-质谱 (LC-MS)，或直接输注 ESI 多反应监测-质谱 (ESI-MRM-MS)11，12来衡量。免疫富集 MRM 检测灵敏度提高 3-4 个数量级，达到低吴/mL 范围13。
与电喷雾质谱相比，质谱速度更快，并且不涉及清洗和重新平衡的 LC 列所以没有携带量和污染的问题，使它更加适合高通量研究14。免疫 MALDI 技术得到了我们的实验室要结合质谱免疫富集的敏感和具体量化的多肽和蛋白质 （基于 proteotypic 多肽定量）15，16 ，17。免疫富集，珠 MALDI 靶板上沉积、 矩阵解的方法添加到珠子，和干燥后板是准备 TOF 质谱分析。从珠肽洗脱不执行作为一个单独的步骤，但亲和绑定物洗脱 MALDI 基质解决方案时将其添加到珠点，从而简化样品制备和样品损失最小化。IMALDI 技术已被应用于各种应用程序18，19，最近经过自动化和用于测量血管紧张素我 (和我) 测定血浆肾素活性 (PRA)20。本议定书将演示用于执行自动化的 iMALDI 测定的过程。以 PRA 测定为例，间天 CVs 的少于 10%已通过自动化，与每一天20744 样品的检测能力。
IMALDI PRA 测定表明这份手稿在不需要蛋白质的消化，作为靶分子 (Ang 我) 是一种肽 1295.7 大分子量。在哪里是必要的蛋白质的消化和肽用作完整的蛋白质代孕其他试剂，选定的肽对 iMALDI 应该是独特和特定于目标蛋白，与大量超过 800 的大，所以它可以很容易区分 c从 MALDI 基质溶液的化学噪声。抗多肽抗体所需的多肽免疫富集。IMALDI 试验测量 PRA 的协议包括四个步骤: 1) 昂代我在人血浆;2） 免疫富集昂我使用抗体包被珠;3） 珠转交 MALDI 靶板和添加矩阵解法;和 4） 信号采集20名 MALDI TOF MS 和数据分析。

<table><tbody><tr><td>Healthy control human plasma</td><td>Bioreclamation</td><td>HMPLEDTA2</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma Aldrich</td><td>09830</td><td></td></tr><tr><td>Ammonium citrate dibasic</td><td>Sigma Aldrich</td><td>09833</td><td></td></tr><tr><td>CHAPS (&gt;=98%)</td><td>Sigma Aldrich</td><td>C9426</td><td></td></tr><tr><td>Albumin from chicken egg white (&gt;98%)</td><td>Sigma Aldrich</td><td>A5503</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma Aldrich</td><td>EDS</td><td></td></tr><tr><td>Alpha-cyano-4-hydroxycinnamic acid</td><td>Sigma Aldrich</td><td>70990</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma Aldrich</td><td>78830</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Thermo Fisher Scientific</td><td>85172</td><td>LC-MS grade</td></tr><tr><td>acetonitrile</td><td>Fluka</td><td>34967</td><td>LC-MS grade</td></tr><tr><td>water</td><td>Fluka</td><td>39253</td><td>LC-MS grade</td></tr><tr><td>acetic acid</td><td>Fluka</td><td>320099</td><td>LC-MS grade</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Roche Diagnostics</td><td>3118169001</td><td></td></tr><tr><td>Dynabeads Protein G magnetic beads</td><td>Thermo Fisher Scientific</td><td>10003D</td><td>2.8 &lt;span class=&quot;font5&quot;&gt;&amp;mu;m, 30 mg/mL&lt;/span&gt;</td></tr><tr><td>anti-Ang I goat polyclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-7419</td><td></td></tr><tr><td>Nat and SIS Ang I</td><td>synthesized at the University of Victoria-Genome BC Proteomics Centre</td><td></td><td></td></tr><tr><td>Automated liquid handling system</td><td>Agilent</td><td>16050-102</td><td>Agilent Bravo robotic workstation</td></tr><tr><td>Magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td>Invitrogen DynaMag-2 magnet</td></tr><tr><td>Tube rotator</td><td>Theromo Scientific</td><td>400110Q</td><td>Labquake Tube Rotator</td></tr><tr><td>Magnet</td><td>Thermo Fisher Scientific</td><td>12027</td><td>DynaMag-96 side skirted magnet</td></tr><tr><td>Magnet</td><td>VP Scientific</td><td>771RM-1</td><td>used to pull the beads to the bottom of the well</td></tr><tr><td>MALDI-TOF</td><td>Bruker</td><td></td><td>Bruker Microflex LRF instrument</td></tr></tbody></table>

peptide and protein quantification using automated immuno-maldi (imaldi)

质谱 (MS) 是量化复杂样品中的蛋白质与优秀测定特异性由于每个目标分子质量电荷比直接检测的最常用的技术之一。然而，基于 MS 的蛋白质组学，像大多数其他分析技术，有偏向于测量高丰度分析物，所以它难实现检测限制的低吴/毫升或 pg/mL 的复杂样品和这许多的浓度范围是疾病相关蛋白等人血浆的关键问题之一。为协助低丰度分析物的检测，免疫富集，只纳入测定浓缩和纯化前 MS 测量，显著提高了检测灵敏度分析物。在这项工作，免疫-Matrix-Assisted 激光解吸电离 (iMALDI) 技术是提出了一种定量的蛋白质和肽的关键问题之一，基于免疫浓缩制成的小珠，紧接着 MALDI 质谱测量不事先洗脱。抗多肽抗体是对磁珠，官能化和培养与样品。洗完后，珠子直接转嫁 MALDI 靶板和信号测定 (MALDI Tof) 仪器 MALDI 时间后矩阵解的方法已被应用于珠。样品制备程序简化相比其他免疫色谱-质谱测定和 MALDI 测量是快速。整个样品制备自动化与液体处理系统，以提高的测定的重现性和更高的吞吐量。在这篇文章，iMALDI 法用于确定肽血管紧张素我 (Ang 我) 用于临床作为读出的血浆肾素活性的筛选原发性醛固酮增多 (PA) 的血浆中的浓度。

李安的测量自动化的 iMALDI 程序我是图 1中所示。关于抗肽磁珠，丰富了目标肽 （内源性肽或从消化蛋白质肽） 和珠子然后转给 MALDI 测量的靶板。整个过程就简化相比需要更多肽洗脱步骤其他免疫色谱-质谱技术。IMALDI 检测自动化允许高通量分析的大量样品间天变异系数 (CV 10 20以下)。代表性的光谱测定人血浆样品中的我<strong class=...

Watch this Scientific Journal Video about Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI at JoVE.com

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

提出了在使用自动的免疫-MALDI (iMALDI) 技术的复杂生物体液蛋白质定量的协议。

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

peptide-and-protein-quantification-using-automated-immuno-maldi-imaldi

Research

JoVE Journal

Chemistry

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

7.9K 次观看.  University of Victoria-Genome BC Proteomics Centre. 提出了在使用自动的免疫-MALDI (iMALDI) 技术的复杂生物体液蛋白质定量的协议。

视频: Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

定量蛋白质组学使用还原二甲基化的稳定同位素标记

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

科研

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

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

生物化学

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI成像帕金森病的神经肽质谱

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

医学

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

使用组合前体同位素标签和同位素标记 （cPILOT） 实现自动样品多路复用

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x - 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments.

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

卡利内克特氏菌中神经肽的多方面质谱研究

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and ...

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome ("surfaceome") in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

摘要

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定量蛋白质组学使用还原二甲基化的稳定同位素标记

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

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

MALDI成像帕金森病的神经肽质谱

通过肽浓缩和质谱检测蛋白质泛化位点检测

使用组合前体同位素标签和同位素标记（cPILOT）实现自动样品多路复用

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

卡利内克特氏菌中神经肽的多方面质谱研究

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

加上质谱肽免疫亲和富集的蛋白质定量

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

摘要

探索更多视频

定量蛋白质组学使用还原二甲基化的稳定同位素标记

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

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

MALDI成像帕金森病的神经肽质谱

通过肽浓缩和质谱检测蛋白质泛化位点检测

使用组合前体同位素标签和同位素标记 （cPILOT） 实现自动样品多路复用

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

卡利内克特氏菌中神经肽的多方面质谱研究

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

加上质谱肽免疫亲和富集的蛋白质定量

使用组合前体同位素标签和同位素标记（cPILOT）实现自动样品多路复用