Uncover New Insights with Post-translational Modification (PTM) Analysis

See how Platinum’s single-molecule resolution overcomes obstacles presented by other PTM analysis methods:

• Integrating Top-Down Mass Spectrometry and Single-Molecule Sequencing for Proteoform Analysis — WEBINAR
  Dr. Michael Caldwell,Scientific Officer at Northwestern Proteomics Center of Excellence, Dr. Meredith Carpenter, Head of Scientific Affairs, QSI and Dr. Kenneth Skinner, Senior Scientific Liaison, QSI September 30, 2024

  Given the numerous sources of protein variation, ranging from biological factors to sample preparation and instrument settings, there is a pressing need for next-generation analytical platforms capable of driving proteoform analysis with complete molecular precision.

  In this webinar, Dr. Michael Caldwell, Scientific Officer at Northwestern Proteomics Center of Excellence, explores top-down mass spectrometry (TD-MS) and Platinum^®.

  Platinum is a benchtop instrument that provides single amino acid resolution of single protein molecules to characterize IL-6, including pyroglutamate (pE), a post-translational modification (PTM) produced via enzymatic and spontaneous cyclization of N-terminal glutamine (Q)/glutamate (E) residues.

  Find out how TD-MS enables intact protein analysis, limiting formation of N-terminal Q/E (MS1). During MS2, analysis of daughter ions reveals modifications and artifactual formation. Platinum employs dye-labeled N-terminal amino acid recognizers that reveal primary structure and variants, and aminopeptidases that expose the next amino acid for recognition until the peptide is sequenced. TD-MS and Platinum detect not only functionally relevant regions of IL-6 primary structure, but also sites prone to pE formation.

  Key Learning Points/Takeaways:

  • Understand how top-down mass spectrometry (TD-MS) and Next-Generation Protein Sequencing can complement each other for comprehensive protein analysis.
  • Explore methods to bridge discovery and targeted protein analysis across different instruments and detection methods.
  • Learn how TD-MS distinguishes between native and artifactual proteoforms.

  Speakers: Michael Caldwell, PhD – Northwestern University, Meredith Carpenter, PhD – QSI and Kenneth Skinner, PhD – QSI

  Date: September 30, 2024

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• Detection and Differentiation of PTMs with Platinum – APP NOTE

  Arginine PTM analysis

  Arginine plays a critical role in protein structure and function and often mediates key interactions between protein binding partners or between proteins and DNA. The two most common arginine PTMs are citrullination and dimethylation, which respectively alter arginine into citrulline or ADMA and SDMA.

  

  ADMA and SDMA isomers share structural similarities and an identical mass, limiting their identification through traditional mass spectrometry. Likewise, determining arginine to citrulline results in a negligible mass increase. Another common method for PTM detection is antibody-based immunoassay ELISA, but only a few antibodies against arginine PTMs are commercially available, restricting its application.

  Direct detection of arginine PTMs in proteins with Platinum

  With Platinum’s single-molecule resolution and sensitivity, you can detect and differentiate arginine PTMs accurately. Peptide fragments are probed in real-time by fluorescently labeled recognizers, designed to bind on and off to their cognate N-terminal amino acid (NAA), while aminopeptidases in solution remove analyzed NAAs to expose subsequent amino acids for further recognition.
  Fluorescence lifetime, intensity, and kinetic data are collected in real-time and analyzed to determine amino acid sequence and PTM content. This novel single-molecule sequencing approach can overcome the challenges facing conventional methods through unparalleled amino acid detection.

  Using Quantum-Si’s single-molecule protein sequencing, we demonstrated how to detect citrulline and how to differentiate between dimethylated arginine residues efficiently.

  See the Data

  Platinum Can Detect and Differentiate PTMs – Download the application note to see how the process works and the PTM analysis that Platinum® delivers.

• Beyond the Genome: Identification of Protein Sequence and PTMs with Quantum-Si’s Next- Generation Protein Sequencer™ Platinum® – POSTER

  To demonstrate the versatility of Platinum and the use of kinetic signatures, we sequenced various types of samples, including mixtures of recombinant proteins, peptides with post-translational modifications (PTMs), proteins immunoprecipitated from human serum, and proteins isolated from human serum via fractionation with SDS-PAGE. The resulting peptides generated distinct kinetic signatures aligned to their respective sequences, highlighting the efficacy of Quantum-Si’s sequencing platform in analyzing multi-protein mixtures at reduced input concentrations.

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• Beyond the Genome: Unraveling Protein Variability with Quantum-Si’s Next-Generation Protein Sequencing™ Technology – POSTER

  Protein sequencing is a groundbreaking advancement in proteomics that augments genomics and transcriptomics research by providing crucial insights into the functional proteins encoded by the genome. Protein sequencing offers a more complete understanding of cellular processes and disease mechanisms by detecting changes at the protein level, such as post-translational modifications (PTMs), which cannot be captured by genomics data alone. Next-Generation Protein Sequencing™ (NGPS) on Platinum® enables researchers to identify and characterize proteins with single-molecule resolution in a simple workflow and on a benchtop instrument.

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