Product ion chromatograms, visualized in Skyline, of peptides from the human ACE2 protein (top) and the SARS-CoV-2 Nucleocapsid protein (bottom), derived from virally-infected A549 cells. Using a standard data-dependent analysis (DDA) LCMS/MS method (left panels) results in no signal or poor peak shape due to instrument duty cycle. A Stable Isotope Standard Assisted Scanning (SISAS) method (right panels) results in consistently detected peptides with quality peak shapes resulting in confident peptide and protein quantitaton.
SignalScan™ LCMS Analysis. SignalScan™ uses a stable isotope standard assisted scanning (SISAS) principle to quantitate targeted peptides without retention time scheduling and with greater sensitivity than untargeted DDA approaches. The heavy standard and the matching endogenous peptide co-elute in the chromatogram but are resolved in the MS1 scan by a mass difference of +8 or +10Da. As peptides enter the mass spectrometer, only MS1 precursor scans are performed until an m/z matching one of the heavy SIS peptides is found. Then a fast, low-resolution MS2 scan event fragments the SIS precursor ion and matches its most abundant fragments to a list of known values. This confirms the identity of the SIS peptide and triggers analysis of the matching endogenous peptide. The light peptide is fragmented and a series of MS2 scans are collected with parameters optimized for high-resolution quantitation. Mutliple fragment intensities are monitored as the peptide elutes, just as in a parallel-reaction-monitoring experiment. The areas under the peak curves are integrated in Skyline and the relative area changes can be calculated for each treatment condition.
SignalScan™ Workflow Steps. The SignalScan™ Peptide Mix (SARS-CoV-2) is compatible with any human sourced biological material. Extract proteins from the tissue and digest them into peptides using any standard trypsin-based protocol. Spike in 1 pmol of the heavy Stable Isotope Standard (SIS) peptides provided prior to LC-MS/MS analysis.
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.com/www/zip/proteomics/signalscan-peptide-mix.zip.
This product is stable for 12 months when stored at -20ºC. Aliquot to avoid multiple freeze/thaw cycles.
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).
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