This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

The authors have nothing to disclose.

绝对蛋白定量是生物医学应用，如生物标志物验证和信号转导通路造型非常多元化至关重要。近日，采用LC-SRM针对性的蛋白质组学得益于改进众多技术，包括肽标准配制，HPLC，QQQ-MS和LC-SRM数据分析。因此，它已成为一个强大的替代免疫。免疫测定可以是非常敏感的，高通量的，但开发强大的免疫测定可以是非常具有挑战性的，因为免疫测定可以是易受交叉反应性和/或干扰，不符合细胞/组织裂解/...

使用质谱（MS）蛋白质组实验可以设计为使用非靶向（散弹枪）或有针对性的方法。发现蛋白质组学通常依赖于自下而上猎枪质谱，或者通过使用一个传统的数据依赖性获取模式，或者通过使用最近开发的与数据无关的技术之一（ 例如 ，MS E，小水）1,2-。鸟枪蛋白质组学是一个功能强大的工具，高通量肽鉴定和相对定量，但它通常是不适合的绝对定量或用于靶向蛋白质的小，定义集（〜数万）。最常用于目标蛋白质组的MS法被选择，因为它的高灵敏度，速度和动态范围3-5的反应监测（SRM）。替代品SRM包括平行反应监测，这需要高分辨率，全MS扫描6的优势。 SRM是使用纳米流逆向工程＆通常进行SED相高效液相色谱法（纳米RP-LC）仪器耦合到纳米电喷雾附着到三重四极质谱仪（QQQ-MS）（纳米ESI）离子源。在典型的实验中，样品的蛋白质被蛋白水解消化，所得的肽色谱分离，解吸和电离。得到的前体离子M / Z过滤由所述第一四极（Q1）和零散的第二四极（Q2）通过与碰撞气体碰撞它们。所得的片段离子是M / Z-过滤在第三四极（Q3）中，并由倍增电极定量。每种前体离子和碎片离子对被称为过渡，并且每个过渡监视在指定的时间段（停留时间;通常2-50毫秒）。在LC-SRM中，通过转换的预定义列表中QQQ-MS周期（占空比是通常≤3秒），并且每个过渡的色谱产生。 替代战略中独立实体为蛋白质定量通常使用的免疫测定，例如斑点印迹，Western印迹，酶联免疫吸附，抗体微阵列，反相蛋白质微阵列，微流体免疫测定，数字的ELISA，以及基于微球的免疫7。最好的免疫测定可以是显著比LC-SRM更加敏感，并且可以通过免疫测定的样品可以是比LC-SRM 5显著更高。然而，开发免疫测定可以是昂贵的和/或费时的，产生的测定法可以是易受交叉反应性和/或干扰，不符合细胞/组织裂解/均匀化的方法，和/或不适合于复用5,8。其中的一些问题可以通过偶联抗体和基于MS的技术来解决。例如，靶蛋白可以通过免疫前蛋白水解和LC-SRM 9-12富集。可替代地，SISCAPA技术采用对蛋白水解随后免疫沉淀在肽LEVE升13,14。除了​​immunoenrichment策略，高丰度蛋白的免疫耗竭可用于增加LC-SRM灵敏度通过减少干扰由共洗脱被分析物15,16。 基于MS的蛋白质定量可以分为相对和绝对定量，并且也为无标记和稳定同位素标记（ 例如 ，代谢标记，化学标记，以及重标记的蛋白质和肽内标准）。无标记技术可以为相对蛋白定量是有用的，但不适合于精确的绝对定量。通过比较，标记技术具有减少与样品制备和MS方差相关的错误，并且经常被用于相对蛋白定量17。例如，稳定同位素标记蛋白质组（SILAP）标准允许使用潜在生物标志物的相对定量通过人血清18 LC-SRM一个培养的人类细胞制备。精确的绝对蛋白定量由MS需要纯化的，量化的，同位素标记的蛋白质或肽内标准被掺入-成在MS之前的生物样品。重同位素标记的内标准纳入的LC-SRM工作流使已被证明是高度可再现和可转让的实验室16,19之间的绝对定量。 稳定同位素标记的内部标准进行绝对蛋白定量经MS包括肽标准，使用固相合成20，蛋白级联的蛋白酶可切割的肽标准21组成，和全长蛋白质标准22制备。靶蛋白共价修饰和不完整的样品制备（ 即不完整的样品裂解和均质化，和不完全蛋白质溶解，变性，烷基化，蛋白水解和）可以破坏精确的定量。内部protein标准是最不容易受到大多数这些潜在的问题，但是它们通常是最难以制备。另一种方法是分析使用被设计为包括氨基和羧基末端天然侧翼残基的多个内部肽标准每个目标蛋白质。无论哪个内标类型采用的，但是应当掺入-到生物样品在早一个点的样品制备尽可能期间。此外，多个样品制备技术（ 例如 ，不同的变性条件）进行测试。多个正交实验技术（实验交叉验证）的使用是一个可行的战略，以克服最有潜力的量化挑战23-25。 蛋白的LC-SRM量化是一个高度灵活的技术，已在各种各样的应用中被使用。值得注意的是，它已被用于在研究的肽和蛋白质生物标志物临床样品如血清，核心活检，以及细针抽吸5。 LC-SRM也已用于测量蛋白质复合物5,26的化学计量，以检测肉毒神经毒素27，向 ​​信号通路5内量化蛋白磷酸化动力学，并量化改变蛋白质构象28。 我们的实验室使用LC-SRM量化介导的巨噬细胞的趋化，以支持趋途径模拟发展的信号蛋白。该协议的总体方案（ 图1）开始排名暂定目标肽。随后，粗外部肽标准合成并用于开发的LC-SRM测定生物样品的定性分析。如果来源于生物样品的靶肽进行检测，纯化的重标记的内部肽标准进行定量的LC-SRM制备。该协议可以用于交流curately从各种各样的生物样品的定量蛋白质，并支持多种蛋白质目标调查。

<table><tbody><tr><td>Acetonitrile (ACN), LC-MS grade</td><td>Fisher</td><td>A955-1</td><td></td></tr><tr><td>BCA (bicinchoninic acid) protein assay kit</td><td>Fisher</td><td>23235</td><td></td></tr><tr><td>Beads for bead beating, zirconia-silica, 0.1 mm</td><td>BioSpec Products</td><td>11079101z</td><td></td></tr><tr><td>Bestatin hydrochloride</td><td>Sigma</td><td>B8385-10MG</td><td></td></tr><tr><td>Cell culture DMEM (with glucose, without L-glutamine)</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>Cell culture EDTA, 500 mM, pH8</td><td>Gibco</td><td>15575</td><td></td></tr><tr><td>Cell culture fetal bovine serum (FBS)</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Cell culture L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>Cell culture phosphate buffered saline (PBS) pH 7.4</td><td>Gibco</td><td>10010-049</td><td></td></tr><tr><td>Cell culture Trypan Blue viability stain, 0.4% w/v</td><td>Lonza</td><td>17-942E</td><td></td></tr><tr><td>Cellometer Auto T4 cell counter</td><td>Nexcelom Bioscience</td><td>Cellometer Auto T4</td><td></td></tr><tr><td>Cellometer Auto T4 disposable counting chambers</td><td>Nexcelom Bioscience</td><td>CHT4-SD100-014</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>D5545-5G</td><td></td></tr><tr><td>Formic acid, LC-MS grade, ampules</td><td>Fisher</td><td>A117-10X1AMP</td><td></td></tr><tr><td>Hemocytometer, Neubauer-improved, 0.1 mm deep</td><td>Marienfeld-Superior</td><td>0640030</td><td></td></tr><tr><td>HEPES, 1 M, pH 7.2</td><td>Mediatech</td><td>25-060-CI</td><td></td></tr><tr><td>Hydrochloric acid, 37% w/w</td><td>VWR</td><td>BDH3028-2.5LG</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>I1149-5G</td><td></td></tr><tr><td>Laser Based Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-2000</td><td></td></tr><tr><td>LC coated silica capillary, 50 &amp;micro;m id</td><td>Polymicro Technologies</td><td>1068150017</td><td></td></tr><tr><td>LC vial, autosampler, 12 mm x 32 mm polypropylene</td><td>SUN SRI</td><td>200-268</td><td></td></tr><tr><td>LC vial screw cap, autosampler, pre-slit PTFE/silicone</td><td>SUN SRI</td><td>500-061</td><td></td></tr><tr><td>Luciferase, from Photinus pyralis</td><td>Sigma</td><td>L9506-1MG</td><td></td></tr><tr><td>Pepstatin A</td><td>EMD Millipore</td><td>516481-25MG</td><td></td></tr><tr><td>pH strips colorpHast (pH 0.0-6.0)</td><td>EMD Chemicals</td><td>9586-1</td><td></td></tr><tr><td>PhosStop phosphatase inhibitor cocktail</td><td>Roche</td><td>04906837001</td><td></td></tr><tr><td>RapiGest SF</td><td>Waters</td><td>186001861</td><td></td></tr><tr><td>Sep-Pak SPE, C18 1 ml 100 mg cartridge</td><td>Waters</td><td>WAT023590</td><td></td></tr><tr><td>Sep-Pak SPE, extraction manifold, 20 position</td><td>Waters</td><td>WAT200609</td><td></td></tr><tr><td>Sep-Pak SPE, flat-surfaced rubber bulb</td><td>Fisher</td><td>03-448-25</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher</td><td>S318-500</td><td></td></tr><tr><td>SpeedVac vacuum concentrator</td><td>Fisher</td><td>SPD111V</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA), LC-MS grade</td><td>Fisher</td><td>A116-50</td><td></td></tr><tr><td>Trypsin, sequencing grade, modified</td><td>Promega</td><td>V5113</td><td></td></tr><tr><td>Tube decapper for Micronic tubes</td><td>USA Scientific</td><td>1765-4000</td><td></td></tr><tr><td>Tubes, 2 ml microcentrifuge, o-ring screw-cap, sterile</td><td>Sarstedt</td><td>72.694.006</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U0631-500g</td><td></td></tr><tr><td>Water, LC-MS grade</td><td>Fisher</td><td>W6-1</td><td></td></tr></tbody></table>

selected reaction monitoring mass spectrometry for absolute protein quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

的信号转导通路预测计算模型的开发是系统生物学53的基本目标之一。不幸的是，即使对于信令已被广泛研究，并具有高的临床意义的途径，它仍然是一般不可能定量预测响应于扰动通路行为（ 例如 ，这是真实的MAPK / ERK通路54）。近日，一项调查采用有针对性的蛋白质组学，转录和计算机建模和仿真研究小鼠巨噬细胞的趋化信号通路56。调查的重点是RAW 264.7细胞（?...

Watch this Scientific Journal Video about Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification at JoVE.com

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

selected-reaction-monitoring-mass-spectrometry-for-absolute-protein

Research

JoVE Journal

Biology

17.0K 次观看.  National Institutes of Health. This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

视频: Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

科研

生物化学

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

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 ...

化学

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 ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent ...

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

基于靶向质谱的低丰度蛋白质蛋白分析中的微量采样

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

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.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

生物学

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

组织样品质谱基猎枪蛋白造影学的综合工作流程

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.