Revision 3
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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Product Information

Storage

This product is stable for 12 months when stored at -20ºC. Aliquot to avoid multiple freeze/thaw cycles.

Product Description

The SignalScan™ Peptide Mix (SARS-CoV-2) enables targeted analysis of selected peptides in human-sourced cells or tissues. Peptides and phosphopeptides derived from SARS-CoV-2 spike and nucleocapsid proteins as well as nine human host response proteins are included. This mixture contains 96 pmol each of 25 heavy-labeled (13C, 15N) Stable Isotope Standard (SIS) peptides, quantified by amino acid analysis (AAA). Inclusion of SIS peptides enhances the sensitivity of the LCMS assay by acting as a trigger for targeted MS/MS analysis. Targeted assays ensure that peptides present in the biological sample of interest are consistently observed and sensitivity is maximized. SIS-assisted scanning provides the sensitivity of a parallel reaction monitoring (PRM) assay without use of rigid retention time windows. Full peptide list, detailed protocol, and files for method set-up and data analysis are available for download at http://media.cellsignal.cn/www/zip/proteomics/signalscan-peptide-mix.zip.

Background

The cause of the COVID-19 pandemic is a novel and highly pathogenic coronavirus, termed SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2). SARS-CoV-2 is a member of the Coronaviridae family of viruses (1). The genome of SARS-CoV-2 is similar to other coronaviruses, and is comprised of four key structural proteins: S, the spike protein, E, the envelope protein, M, the membrane protein, and N, the nucleocapsid protein (2). Coronavirus spike proteins are class I fusion proteins and harbor an ectodomain, a transmembrane domain, and an intracellular tail (3,4). The highly glycosylated ectodomain projects from the viral envelope surface and facilitates attachment and fusion with the host cell plasma membrane. The ectodomain can be further subdivided into host receptor-binding domain (RBD) (S1) and membrane-fusion (S2) subunits, which are produced upon proteolysis by host proteases at S1/S2 and S2’ sites. S1 and S2 subunits remain associated after cleavage and assemble into crown-like homotrimers (2,4). In humans, both SARS-CoV and SARS-CoV-2 spike proteins utilize the angiotensin-converting enzyme 2 (ACE2) protein as a receptor for cellular entry (5-7). The cell’s subsequent host response to SARS-CoV-2 infection involves complex signaling cascades, observable through perturbations in protein phosphorylation patterns (8,9) that lead to interferon and cytokine production (10).

  1. Zhou, P. et al. (2020) Nature 579, 270-273.
  2. Tortorici, M.A. and Veesler, D. (2019) Adv Virus Res 105, 93-116.
  3. Li, F. et al. (2006) J Virol 80, 6794-800.
  4. Li, F. (2016) Annu Rev Virol 3, 237-261.
  5. Shang, J. et al. (2020) Nature 581, 221-224.
  6. Wrapp, D. et al. (2020) Science 367, 1260-1263.
  7. Yan, R. et al. (2020) Science 367, 1444-1448.
  8. Davidson, A.D. et al. (2020) Genome Med 12, 68.
  9. Bouhaddou, M. et al. (2020) Cell 182, 685-712.e19.
  10. Blanco-Melo, D. et al. (2020) Cell 181, 1036-1045.e9.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalScan is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

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Revision 3
#23733

SignalScan Peptide Mix (SARS-CoV-2)

SignalScan™ Peptide Mix (SARS-CoV-2): Image 1 Expand Image
在 Skyline 中可视化的人 ACE2 蛋白(上图)和 SARS-CoV-2 核衣壳蛋白(下图)衍生自病毒感染的 A549 细胞的肽的产物离子色谱图。使用标准的数据相关分析(DDA)LCMS/MS 方法(左图),由于仪器的工作循环而导致没有信号或峰形差。稳定同位素标准辅助扫描(SISAS)方法(右图)可稳定检测出具有高质量峰形的肽,从而对肽和蛋白质进行定量分析。
SignalScan™ Peptide Mix (SARS-CoV-2): Image 2 Expand Image
SignalScan LCMS 分析。SignalScan 使用稳定的同位素标准辅助扫描(SISAS)原理对目标肽进行定量,而没有保留时间安排,并且比非目标 DDA 方法具有更高的灵敏度。重标准品和相匹配的内源肽在色谱图中共洗脱,但在 MS1 扫描中通过 +8 或 +10Da 的质量差得以分离。 当肽进入质谱仪时,仅进行 MS1 前体扫描,直到找到与重 SIS 肽之一匹配的 m/z。然后,快速、低分辨率的 MS2 扫描事件将 SIS 前驱物离子碎片化,并将其最丰富的碎片与已知值列表匹配。这证实了 SIS 肽的标志并触发了匹配的内源肽的分析。轻肽片段化,并使用针对高分辨率定量优化的参数收集了一系列 MS2 扫描。就像在平行反应监测实验中一样,随着肽洗脱,监测了多个片段强度。峰曲线下的面积已整合到 Skyline 中,并且可以针对每种治疗条件计算相对面积变化。
SignalScan™ Peptide Mix (SARS-CoV-2): Image 3 Expand Image
SignalScan 工作流程步骤。SignalScan Peptide Mix (SARS-CoV-2) 与任何人类来源的生物材料兼容。使用任何基于胰蛋白酶的标准协议从组织中提取蛋白质并将其消化成肽。进样 1 pmol 重质稳定同位素标准(SIS)肽,然后进行 LC-MS/MS 分析。