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Protein Phosphorylation Analysis by Mass Spectrometry

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Protein phosphorylation, a reversible process, is characterized by adding phosphate donated from ATP and removing phosphate from a phosphorylated protein substrate. For more information, please visit: https://www.creative-proteomics.com/services/phosphorylation.htm

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Protein Phosphorylation Analysis by Mass Spectrometry

  1. 1. Creative Proteomics Phosphorylation Analysis by Mass Spectrometry
  2. 2. Protein Phosphorylation Pi Phosphatase Kinase ATP ADP P  A reversible process is characterized by adding phosphate donated from ATP and removing phosphate from a phosphorylated protein substrate.  Protein phosphorylation is catalyzed by protein kinase and phosphatase (PP).
  3. 3. Protein Phosphorylation 1 Serine 84.6% 2 Threonine 11.8% 3 Tyrosine 1% Phospho Ser PhosphoThr PhosphoTyr
  4. 4. Protein Phosphorylation Humphrey, Sean J., David E. James, and Matthias Mann. "Protein phosphorylation: a major switch mechanism for metabolic regulation." Trends in Endocrinology & Metabolism26.12 (2015): 676-687. • Protein phosphorylation plays important roles in many cellular processes, including cell cycle, growth, apoptosis and signal transduction pathways. • Phosphorylation can modulate enzyme activity, alter its affinity to other proteins, and transmit signals through kinase cascades that often are branched and interactive.
  5. 5. Protein Phosphorylation Kinase activity assays Phosphor-specific antibody development Western blot Enzyme-linked immunosorbent assay (ELISA) Intracellular Flow Cytometry and immunocytochemistry / immunohistochemistry (ICC/IHC) 1 2 3 5 4 Methods
  6. 6. Protein Phosphorylation MS analysis plays an important role in phosphorylation analysis Identifying phosphorylation sites from individual proteins and small protein complexes Identifying global phosphorylation sites from whole cell and tissue extracts.
  7. 7. Single protein (protein complex) phosphorylation site mapping 01 02 03 Sample preparation Protein purification Phosphorylated peptides enchment Analysis by LC-MS/MS Ionization: electrospray ionization (ESI), or matrix-assisted laser/desorption ionization (MALDI) Fragmentation:collision-induced dissociation (CID), electron capture dissociation (ECD) and Electron transfer dissociation(ETD) Data analysis Informatics software
  8. 8. Global Analysis of Protein Phosphorylation by Mass Spectrometry The identification of large numbers of phosphoproteins and phosphosites Improvements in mass spectrometry The advent of methods for enrichment of phosphoproteins • From hundreds of studies, more than 300,000 phosphorylation sites have been identified across a multitude of both prokaryotic and eukaryotic species, with more than 200,000 of these coming from mammals alone. • Greater than 95% of these sites have been identified using MS-based phosphoproteomics.
  9. 9. Global Analysis of Protein Phosphorylation by Mass Spectrometry Breitkopf, Susanne B., and John M. Asara. "Determining in vivo phosphorylation sites using mass spectrometry." Current protocols in molecular biology (2012): 18-19.
  10. 10. Phosphorylation Analysis Creative Proteomics has established a highly sensitive HPLC- MS/MS platform that can analyze phosphorylation in multiple samples and in both eukaryotic and prokaryotic organisms. In addition, we have optimized our protocol to enable more fast and sensitive site mapping service for phosphorylation analysis. Our Services
  11. 11. Thanks Web: www.creative-proteomics.com Email: info@creative-proteomics.com

Notas

  • Hello, welcome to watch Creative Proteomics’Video. Today, we are going to introduce the Phosphorylation Analysis by Mass Spectrometry.
  • Protein phosphorylation, a reversible process, is characterized by adding phosphate donated from ATP and removing phosphate from a phosphorylated protein substrate. And the protein phosphorylation is catalyzed by protein kinase and phosphatase
  • In eukaryotic cells, phosphorylation occurs most commonly on serine, followed by threonine and tyrosine residues, with the reaction of each phosphorylated in mammalian cells found by phosphorylated in mammalian cells found by phosphoproteomics studies to be around 84%,15%, and <1%.
  • Protein phosphorylation plays important roles in many cellular processes, including cell cycle, growth, apoptosis and signal transduction pathways. In addition, phosphorylation can modulate enzyme activity, alter its affinity to other proteins, and transmit signals through kinase cascades that often are branched and interactive.
  • In order to understand these mechanismas, it is necessary to analyze protein phosphorylation. There are many methods for phosphorylation analysis, including kinase activity assays, phosphor-specific antibody development, western blot, enzyme-linked immunosorbent assay, as well as Intracellular Flow Cytometry and immunocytochemistry / immunohistochemistry.
  • In addition to these methods, MS analysis plays an important role in phosphorylation analysis. There are two common strategies for identifying protein phosphorylation sites through bottom-up proteomics, including identifying phosphorylation sites from individual proteins and small protein complexes, and Identifying global phosphorylation sites from whole cell and tissue extracts.
  • Identifying phosphorylation sites of a specific protein of interest is important for comprehensively study the functional role of phosphorylation from the protein. There are several steps, including Sample preparation, Analysis by tandem mass spectrometry, and data analysis. For Sample preparation, because of the low stoichiometric level of phosphorylated peptides compared to unmodified peptides, there is a need to purify as much protein as possible. In addition, to enrich phosphorylated peptides, there are several methods available, including metal ion affinity chromatography and titanium dioxide beads. For mass spectrometry analysis, the peptides can be introduced into the mass spectrometer through liquid chromatography coupled to electrospray ionization, or matrix-assisted laser desorption ionization. Peptides are most commonly sequenced by tandem mass spectrometry using collision-induced dissociation. Electron capture dissociation and Electron transfer dissociation can also be applied for phosphorylation analysis. And informatics software is necessary to identify peptide sequences and their phosphorylated counterparts.
  • The advent of methods for enrichment of phosphoproteins in conjunction with improvements in mass spectrometry has resulted in the identification of large numbers of phosphoproteins and phosphosites. With the advancement of MS instrumentation that allows for the rapid and sensitive sequencing of peptides and the concurrent development of phosphopeptide enrichment strategies, we now have the ability to analyze changes in protein phosphorylation at an unprecedented scale.

    From hundreds of studies, more than 300,000 phosphorylation sites have been identified across a multitude of both prokaryotic and eukaryotic species, with more than 200,000 of these coming from mammals alone. Greater than 95% of these sites have been identified using MS-based phosphoproteomics.
  • Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time-consuming. It has several strategies.
    This strategy involves digesting the whole cell lysate, followed by peptide fractionation by strong cation exchange chromatography (SCX), phosphopeptide enrichment by IMAC or TiO2.
    In this strategy, the protein lysate can be fractionated by SDS-PAGE, followed by digestion, phosphopeptide enrichment and LC-MS/MS.
    In addition, one can also IP only phospho-tyrosine peptides using a pTyr antibody followed by LC-MS/MS.
  • Creative Proteomics has established a highly sensitive HPLC-MS/MS platform that can analyze acetylation in multiple samples and in both eukaryotic and prokaryotic organisms. In addition, we have optimized our protocol to enable more fast and sensitive site mapping service for acetylation analysis.
  • Thanks for watching our video. At creative proteomics, we provide the most reliable serivices. If you have any questions or specific requirements. Please do not hesitate to contact us. We are very glad to cooperate with you.

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