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PROTEIN-B
PROTEIN-C
PROTEIN-A
Anil Gattani
P-2021
• Protein structure profiling and protein-protein
interactions is crucial to define the protein functions in
biological systems
• Protein interactions is fundamental to understanding
biological processes
• A single protein may interact with many other
molecules
• Protein interactions are often quite transient in nature
or may involve large multiprotein complexes
• In PDB 128962 Protein structure entries are
available
X ray crystallography
NMR spectroscopy
Trifunctional cross linker
Mass Spectrometry – Based Approaches
• Mass spectrometry (MS) was pioneered by the work of
J. J. Thomson- 1906
• The Nobel Prize in Chemistry in 2002—MALDI—Tanaka
and ESI-- Fenn
• MS evolved as analytical technique in protein science
• Mass spectrometry can also be employed for the
analysis of noncovalent protein complexes
• Chemical cross-linking with MS represent an approach
for the structural characterization of proteins
Chemical cross linking (CX)
• Chemically joining two or more molecules by a covalent
bond
• For determination of near neighbor relationships, 3-D
structures of proteins, and molecular associations in cell
membranes
• Neither a throughput technique nor provide information
about strength of interaction
• Allow higher resolution data where PPI mapped to specific
domains or Amino acid
• Allows non covalent PPI to be captured into long lived
covalent complexes
Functional group--
amine-, sulfhydryl-, or
photo-reactive
Linker/Spacer arm– ruler to measure length
Functional groups of cross linking reagents
• Amine reactive chemistries
N -hydroxysuccinimide
• Sulfhydryl-reactive chemistries
• Carbonyl-/Glyco-reactive chemistry
• Carboxyl-reactive chemistry
• Nonspecific chemistries
Aryl azides
N-hydroxysuccinimide (NHS) esters have been the most
frequently-used reactive group
• Form stable amide linkage to the primary amines in
proteins at physiological condition (pH 7.0–7.5), which
is important for in vivo labeling
• the most common targets for labeling proteins are the
primary amine groups, which are present on the large
majority of proteins due to the high frequency of
lysine residues in most proteins
• NHS ester reaction has very rapid reaction rates with
primary amines
Trifunctional cross linker
Trifunctional Crosslinkers
• Three different reactive or complexing groups per
molecule
• Initial attempts at producing trifunctional reagents
used biocytin as the core compound
Wedekind et al. (1989)
Trifunctional cross linker
Ed Fujimoto
Label transfer
Trifunctional cross linker
Frei et al. (2012)
M. U. Mayer et al. 2009
Targeted Releasable Affinity Probe (TRAP) for
In Vivo Photocrosslinking
Tang et al. (2005)
Protein Interaction Reporter
• Protein Interaction Reporter (PIR), which can be
cleaved directly in the mass spectrometer to
release the two individual peptide chains
• The labile bonds in the cross-link can be
specifically cleaved in situ to release two intact
peptide chains
• Each peptide chain can then be sequenced
separately and identified either by MSMS
fragmentation or accurate mass measurement
Trifunctional cross linker
Tan et al. (2016)
Trifunctional cross linker
Trifunctional cross linker
COMPUTER SOFTWARE FOR DATA ANALYSIS
• Automated Spectrum Assignment Program (ASAP)
• GPMAW program (General Protein/Mass Analysis for
Windows)
• MassMatrix
• xQuest/xProphet
• CrossWork
• xComb
• StavroX
• pLink
Trifunctional cross linker
Types of Databases
• The majority of cross-linking studies is currently
performed with amine-reactive cross-linkers, which are
employed as mixtures of light and heavy derivatives to
facilitate the identification of cross-linked peptides
• Chemical cross-linking/mass spectrometry (MS) can be
used in concert with other low-resolution methods for
protein structure analysis to obtain complementary
structural information on protein assemblies
• The current bottleneck in chemical cross-linking/MS is
the availability of data analysis tools for a fully
automated workflow
References
Andrea Sinz (2014). The advancement of chemical cross-linking and mass
spectrometry for structural proteomics: from single proteins to protein interaction
networks. Expert Rev. Proteomics 11(6)
Tan et al. eLife 2016;5:e12509. DOI: 10.7554/eLife.12509
Preston, GW and Wilson, AJ (2013). Photo-induced covalent cross-linking for the
analysis of biomolecular interactions. Chem. Soc. Rev.,42
Xiaobin Xu (2015). Chemical Cross-linking Mass Spectrometry for Profiling
Protein Structures and Protein-Protein Interactions. Proteomics Bioinform, 8:12
Suchanek,M, Redzikowaska,A and Thiele,C (2005). Photo leucine and photo
methioninealoow identification of protein protein interaction in living cells.
Nature Methods, DOI:10.1038/NEMTH752
Leitner et al (2014). Chemical cross-linking/mass spectrometry targeting acidic
residues in proteins and protein complexes. PNAS :111
Trifunctional cross linker

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Trifunctional cross linker

  • 2. • Protein structure profiling and protein-protein interactions is crucial to define the protein functions in biological systems • Protein interactions is fundamental to understanding biological processes • A single protein may interact with many other molecules • Protein interactions are often quite transient in nature or may involve large multiprotein complexes
  • 3. • In PDB 128962 Protein structure entries are available X ray crystallography NMR spectroscopy
  • 5. Mass Spectrometry – Based Approaches • Mass spectrometry (MS) was pioneered by the work of J. J. Thomson- 1906 • The Nobel Prize in Chemistry in 2002—MALDI—Tanaka and ESI-- Fenn • MS evolved as analytical technique in protein science • Mass spectrometry can also be employed for the analysis of noncovalent protein complexes • Chemical cross-linking with MS represent an approach for the structural characterization of proteins
  • 6. Chemical cross linking (CX) • Chemically joining two or more molecules by a covalent bond • For determination of near neighbor relationships, 3-D structures of proteins, and molecular associations in cell membranes • Neither a throughput technique nor provide information about strength of interaction • Allow higher resolution data where PPI mapped to specific domains or Amino acid • Allows non covalent PPI to be captured into long lived covalent complexes
  • 7. Functional group-- amine-, sulfhydryl-, or photo-reactive Linker/Spacer arm– ruler to measure length
  • 8. Functional groups of cross linking reagents • Amine reactive chemistries N -hydroxysuccinimide
  • 9. • Sulfhydryl-reactive chemistries • Carbonyl-/Glyco-reactive chemistry
  • 10. • Carboxyl-reactive chemistry • Nonspecific chemistries Aryl azides
  • 11. N-hydroxysuccinimide (NHS) esters have been the most frequently-used reactive group • Form stable amide linkage to the primary amines in proteins at physiological condition (pH 7.0–7.5), which is important for in vivo labeling • the most common targets for labeling proteins are the primary amine groups, which are present on the large majority of proteins due to the high frequency of lysine residues in most proteins • NHS ester reaction has very rapid reaction rates with primary amines
  • 13. Trifunctional Crosslinkers • Three different reactive or complexing groups per molecule • Initial attempts at producing trifunctional reagents used biocytin as the core compound Wedekind et al. (1989)
  • 18. Frei et al. (2012)
  • 19. M. U. Mayer et al. 2009 Targeted Releasable Affinity Probe (TRAP) for In Vivo Photocrosslinking
  • 20. Tang et al. (2005) Protein Interaction Reporter
  • 21. • Protein Interaction Reporter (PIR), which can be cleaved directly in the mass spectrometer to release the two individual peptide chains • The labile bonds in the cross-link can be specifically cleaved in situ to release two intact peptide chains • Each peptide chain can then be sequenced separately and identified either by MSMS fragmentation or accurate mass measurement
  • 23. Tan et al. (2016)
  • 26. COMPUTER SOFTWARE FOR DATA ANALYSIS • Automated Spectrum Assignment Program (ASAP) • GPMAW program (General Protein/Mass Analysis for Windows) • MassMatrix • xQuest/xProphet • CrossWork • xComb • StavroX • pLink
  • 29. • The majority of cross-linking studies is currently performed with amine-reactive cross-linkers, which are employed as mixtures of light and heavy derivatives to facilitate the identification of cross-linked peptides • Chemical cross-linking/mass spectrometry (MS) can be used in concert with other low-resolution methods for protein structure analysis to obtain complementary structural information on protein assemblies • The current bottleneck in chemical cross-linking/MS is the availability of data analysis tools for a fully automated workflow
  • 30. References Andrea Sinz (2014). The advancement of chemical cross-linking and mass spectrometry for structural proteomics: from single proteins to protein interaction networks. Expert Rev. Proteomics 11(6) Tan et al. eLife 2016;5:e12509. DOI: 10.7554/eLife.12509 Preston, GW and Wilson, AJ (2013). Photo-induced covalent cross-linking for the analysis of biomolecular interactions. Chem. Soc. Rev.,42 Xiaobin Xu (2015). Chemical Cross-linking Mass Spectrometry for Profiling Protein Structures and Protein-Protein Interactions. Proteomics Bioinform, 8:12 Suchanek,M, Redzikowaska,A and Thiele,C (2005). Photo leucine and photo methioninealoow identification of protein protein interaction in living cells. Nature Methods, DOI:10.1038/NEMTH752 Leitner et al (2014). Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes. PNAS :111