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Strategies for Post-translational Modification (PTM)

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Mass spectrometry (MS) is the suitable method for the analysis of protein modifications because it can provide universal information about protein modifications without a priori knowledge and locating the sites of modification.
If you are interested in our services, please visit: https://www.creative-proteomics.com/services/protein-post-translational-modification-analysis.htm

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Strategies for Post-translational Modification (PTM)

  1. 1. Strategies for Post-Translational Modification (PTM) Presented by Creative Proteomics
  2. 2. Introduction Post-translational modification (PTM) refers to the modification that occurs on a protein after translation catalyzed by enzymes. PTMs play important roles in cell biology, including cell signaling, cell structure, DNA modification, and so on. There are various types of PTMs, including phosphorylation, acetylation, methylation, glycosylation, and so on. 01 02 03
  3. 3. Introduction 01 Traditional strategies, like radioactive labeling and Western blotting, can be specific and relatively quantitative, but they require prior knowledge of the modification type and are limited by antibody availability and specificity. 02 Mass spectrometry (MS) is the suitable method for the analysis of protein modifications because it can provide universal information about protein modifications without a priori knowledge and locating the sites of modification. 03 There are several strategies for PTM identification, including bottom-up, top-down, and middle-down approaches. Doll, Sophia, and Alma L. Burlingame. "Mass spectrometry-based detection and assignment of protein posttranslational modifications." ACS chemical biology 10.1 (2014): 63-71.
  4. 4. Bottom-up Strategy In bottom-up strategy, a protein is typically digested with an enzyme (like trypsin) into small peptides in gel or in solution. These peptides will be detected by mass spectrometer and modification can be mapped in the recovered peptides. Zhang, Han, and Ying Ge. "Comprehensive analysis of protein modifications by top-down mass spectrometry." Circulation: Genomic and Precision Medicine 4.6 (2011): 711-711. Advantages Limitations Has the higher sensitivity than top-down method Low percentage coverage of the protein sequence Increased overall complexity of the sample The connection between modifications on disparate portions of a protein can be lost
  5. 5. Top-down Strategy Zhang, Han, and Ying Ge. "Comprehensive analysis of protein modifications by top-down mass spectrometry." Circulation: Genomic and Precision Medicine 4.6 (2011): 711-711. • The whole protein is directly analyzed in the MS without digestion, so the full information of the modification state can be revealed. • It can universally detect all the existing modifications, including PTMs (ie, phosphorylation, proteolysis, and acetylation) and sequence variants (ie, mutants, alternatively spliced isoforms, and amino acid polymorphisms) simultaneously in one spectrum without a priori knowledge.
  6. 6. Top-down Strategy Zhang, Han, and Ying Ge. "Comprehensive analysis of protein modifications by top-down mass spectrometry." Circulation: Genomic and Precision Medicine 4.6 (2011): 711-711. Top The molecular weight of an intact protein is first measured and compares it with the calculated value based on the DNA-predicted protein sequence, which can reveal modifications in the protein sequence globally. A specific modified form of interest can be directly isolated in the mass spectrometer and subsequently fragmented by tandem MS for reliable mapping of the modification sites. Down
  7. 7. Middle-down Strategy Switzar L, Giera M, Niessen W M A. Protein digestion: an overview of the available techniques and recent developments[J]. Journal of proteome research, 2013, 12(3): 1067-1077. • The middle-down approach can be considered as a variant of the top-down approach. • The proteins are needed to soft proteolysis to obtain the large peptides usually by using AspN and GluC. The peptides are then sequenced by tandem mass spectrometry using technologies similar to top-down. • For example, the middle-down strategy can be used for histone analysis, in which proteins are commonly digested into peptides in the 3 to 9 kDa range. Largely preserve the combinatorial modifications of the histone tail, while approaching the sensitivity of the bottom-up approach.
  8. 8. In MS-based PTM analysis, it is important to produce peptide fragmentation information for high confidence sequence identification and site localization of PTMs. There are several fragmentation strategies available. MS Fragmentation Strategy Collision induced dissociation (CID) Higher energy collisional dissociation (HCD) Electron capture dissociation (ECD) Electron transfer dissociation (ETD)
  9. 9. Collision Induced Dissociation (CID) Collision gas Precursor ion Fragment ion Neutral lost  CID, also known as collisionally activated dissociation (CAD), is the most common and widely used unimolecular dissociation method.  In CID, ions collide with neutral molecules to be positively charged.  The CID technique is generally more effective for small and low- charge state peptides  CID is not suitable for fragmentation of intact proteins and peptides with labile post-translational modifications, like phosphorylation.
  10. 10. Higher Energy Collisional Dissociation (HCD) • HCD, known as the beam-type CID, is a CID technique specific to the Orbitrap mass analyzer. • Compared to CID, HCD is featured with higher activation energy and shorter activation time. • HCD can generate b- and y-type fragment ions. While the higher energy for HCD leads to a predominance of y-ions; b-ions can be further fragmented to a-ions or smaller species
  11. 11. Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD)  Electron-based fragmentation methods, achieve fragmentation through neutralization of backbone protonation sites with thermal electrons (ECD) or radical anions (ETD).  They can generate c and z- type fragment ions without losing the PTM localization information.  ECD can only be implemented on Fourier transform ion cyclotron resonance (FTICR) MS instruments, ETD can be implemented on high resolution tandem MS instruments.
  12. 12. Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD) • ECD and ETD perform better with highly charged state analytes, while CID is more efficient with low-charge state peptides. • ECD and ETD are more suitable for detecting unstable PTMs for the reason that peptide backbone fragmentation is virtually independent of the amino acid sequence. In addition, neutral losses are reduced, and O-GlcNAc elimination does not happen.  For highly charged state analytes and unstable PTMs
  13. 13. Our Services Glycosylation analysis Phosphorylation analysis Ubiquitination analysis Methylation analysis Nitrosylation analysis Acetylation analysis With years’ experience in advanced experiment equipment, Creative Proteomics can provide a variety of PTM services to assist your scientific research.
  14. 14. Thanks for Watching Web:www.creative-proteomics.com Email: info@creative-proteomics.com Creative Proteomics

Notas

  • Post-translational modification (PTM) refers to the modification that occurs on a protein after translation catalyzed by enzymes. There are various types of PTMs, including phosphorylation, acetylation, methylation, glycosylation, and so on. PTMs play important roles in cell biology, including cell signaling, cell structure, DNA modification, and so on.
  • For the analysis of protein modification, traditional strategies, like radioactive labeling and Western blotting, can be specific and relatively quantitative, but they require prior knowledge of the modification type and are limited by antibody availability and specificity. Mass spectrometry (MS) is the suitable method for the analysis of protein modifications because it can provide universal information about protein modifications without a priori knowledge and locating the sites of modification. There are several strategies for PTM identification, including bottom-up, top-down, and middle-down approaches.
  • The bottom-up strategy for PTM identification is the traditional proteomic approach. In bottom-up strategy, a protein is typically digested with an enzyme (like trypsin) into small peptides in gel or in solution. These peptides will be detected by mass spectrometer and modification can be mapped in the recovered peptides. Bottom-up strategy has the higher sensitivity than top-down method. However, there are some intrinsic limitations in characterizing protein modifications for the bottom-up strategies. The first one is low percentage coverage of the protein sequence, which leads to the modification status of the unrecovered sequence portion remains unknown. In addition, the overall complexity of the sample is increased due to plentiful small peptide components by protein digestion. Moreover, the connection between modifications on disparate portions of a protein can be lost because the typical peptides from tryptic digestion contain only ≈5 to 20 amino acids.
  • Top-down proteomics is becoming a powerful strategy for analysis of protein modifications. For the top-down strategy, the whole protein is directly analyzed in the MS without digestion, so the full information of the modification state can be revealed. It can universally detect all the existing modifications, including PTMs (ie, phosphorylation, proteolysis, and acetylation) and sequence variants (ie, mutants, alternatively spliced isoforms, and amino acid polymorphisms) simultaneously in 1 spectrum without a priori knowledge
  • Top-down proteomics is becoming a powerful strategy for analysis of protein modifications. For the top-down strategy, the whole protein is directly analyzed in the MS without digestion, so the full information of the modification state can be revealed. It can universally detect all the existing modifications, including PTMs (ie, phosphorylation, proteolysis, and acetylation) and sequence variants (ie, mutants, alternatively spliced isoforms, and amino acid polymorphisms) simultaneously in 1 spectrum without a priori knowledge. In this strategy, the molecular weight of an intact protein is first measured and compares it with the calculated value based on the DNA-predicted protein sequence, which can reveal modifications in the protein sequence globally. A specific modified form of interest can be directly isolated in the mass spectrometer and subsequently fragmented by tandem MS for reliable mapping of the modification sites. Although top-down strategy has many advantages for protein modification analysis, there are still some challenges needed to be resolved, including protein solubility, sensitivity and detection limit, as well as the requirement of high-end instruments.
  • Middle-down proteomics has recently emerged as high throughput strategy to define PTM co-existence frequency. The middle-down approach can be considered as a variant of the top-down approach. In this strategy, the proteins are needed to soft proteolysis to obtain the large peptides usually by using AspN and GluC. The peptides are then sequenced by tandem mass spectrometry using technologies similar to top-down. For example, the middle-down strategy can be used for histone analysis, in which proteins are commonly digested into peptides in the 3 to 9 kDa range. The middle-down approaches largely preserve the combinatorial modifications of the histone tail, while approaching the sensitivity of the bottom-up approach.
  • In MS-based PTM analysis, it is important to produce peptide fragmentation information for high confidence sequence identification and site localization of PTMs. There are several fragmentation strategies available, including collision induced dissociation (CID), higher energy collisional dissociation (HCD), electron capture dissociation (ECD), and electron transfer dissociation (ETD).
  • CID, also known as collisionally activated dissociation (CAD), is the most common and widely used unimolecular dissociation method. In CID, ions collide with neutral molecules (eg.) to be positively charged. The CID technique is generally more effective for small and low-charge state peptides. CID is not suitable for fragmentation of intact proteins and peptides with labile post-translational modifications, like phosphorylation.
  • HCD, known as the beam-type CID, is a CID technique specific to the orbitrap mass analyzer. Compared to CID, HCD HCD is featured with higher activation energy and shorter activation time.
  • ECD and ETD, electron-based fragmentation methods, achieve fragmentation through neutralization of backbone protonation sites with thermal electrons (ECD) or radical anions (ETD). In this way, they can generate c and z- type fragment ions without losing the PTM localization information. ECD can only be implemented on Fourier transform ion cyclotron resonance (FTICR) MS instruments, ETD can be implemented on high resolution tandem MS instruments.
  • ECD and ETD perform better with highly charged state analytes, while CID is more efficient with low-charge state peptides. However, ECD and ETD are more suitable for detecting unstable PTMs for the reason that peptide backbone fragmentation is virtually independent of the amino acid sequence, neutral losses are reduced, and O-GlcNAc elimination does not happen.
  • With years’ experience in advanced experiment equipment, Creative Proteomics can provide a variety of PTM services to assist your scientific research. We can provide Phosphorylation analysis, Glycosylation analysis, Methylation analysis, Acetylation analysis, Ubiquitination analysis, and Nitrosylation analysis.
  • Thanks for watching our video. At creative proteomics, we provide the most reliable services. 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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