Characterization of peptide?protein interactions using photoaffinity labeling and LC/MS

Jahn, Olaf; Eckart, Klaus; Tezval, Hossein; Spiess, Joachim

doi:10.1007/s00216-003-2353-8

Cited by 17 publications

(17 citation statements)

References 18 publications

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“…Then the mixture was irradiated with UV-light (400 watt metal vapor lamp, Ultratech, Osram, Germany) on ice for 30 min. To avoid photodamage of the protein UV light with wavelengths below 300 nm was filtered out by a special glass filter plate (B270, Schott, Germany) (21,22). The photoadducts were analyzed by Western blotting using anti-RGS-His 6 or anti-biotin monoclonal antibodies as primary antibodies and a horseradish peroxidaseconjugated goat anti-mouse antibody as secondary antibody.…”

Section: Construction Of Expressionmentioning

confidence: 99%

Molecular Characterization of the Human Cα-formylglycine-generating Enzyme

Preusser-Kunze¹,

Mariappan²,

Schmidt³

et al. 2005

Journal of Biological Chemistry

139

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C␣-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calciumbinding protein containing an N-terminal (residues 86 -168) and a C-terminal (residues 178 -374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the Nterminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocrosslinking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.

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Section: Construction Of Expressionmentioning

confidence: 99%

Molecular Characterization of the Human Cα-formylglycine-generating Enzyme

Preusser-Kunze¹,

Mariappan²,

Schmidt³

et al. 2005

Journal of Biological Chemistry

139

View full text Add to dashboard Cite

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“…www.future-drugs.com 403 regarding protein-protein interactions by combining photoaffinity labeling techniques with MS has grown tremendously [16,18]. By integrating MS with PAL, researchers have not only identified binding partners for a particular ligand or receptor of interest, but also localized and characterized the sites of interaction at a molecular level [19][20][21][22][23][24][25] with far greater efficiency and rapidity than could be achieved with more traditional biophysical techniques.…”

Section: Photoaffinity Labeling With Mass Spectrometry In Structural mentioning

confidence: 99%

“…Jahn and colleagues recently reported the use of optimized liquid chromatography (LC) separation of photolabeled protein-protein complexes in conjunction with multiple ion chromatogram (MIC) on-line ESI-MS to characterize low abundance complexes [18]. Special reversed-phase LC columns compatible with formic acid enabled them to eliminate the ion suppression effects commonly observed with the reversed-phase ion paring agent trifluoroacetic acid (TFA), resulting in a one order of magnitude increase in MS sensitivity.…”

Section: Photoaffinity Labeling With Mass Spectrometry In Structural mentioning

confidence: 99%

“…By integrating MS with PAL, researchers have not only identified binding partners for a particular ligand or receptor of interest, but also localized and characterized the sites of interaction at a molecular level [19][20][21][22][23][24][25] with far greater efficiency and rapidity than could be achieved with more traditional biophysical techniques. At the protein level, comparing the mass of photolabeled protein-protein ligand-receptor complexes with that of unlabeled material can be used to generate information on the stoichiometry of interactions [18]. By proteolytically cleaving and then purifying a protein-protein oligopeptide photoadduct by electrophoresis or chromatography, investigators can then use MS/MS-based peptide sequencing techniques to determine the amino acid sequence of the binding regions of both ligand and receptor protein.…”

mentioning

confidence: 99%

“…By incorporating a deuterium-labeled p-benzoyl-phenylalanine photoreactive group into substance P and using this probe to label the human NK-1 receptor, the investigators used MALDI-MS to discriminate the photolabeled complex based on double peaks separated by the mass difference between the deuterated and nondeuterated species following digestion of the complex and biotin/streptavidin purification. Using this approach, very minute amounts of protein-protein complex in the subpicomolar range can be confidently characterized.Jahn and colleagues recently reported the use of optimized liquid chromatography (LC) separation of photolabeled protein-protein complexes in conjunction with multiple ion chromatogram (MIC) on-line ESI-MS to characterize low abundance complexes [18]. Special reversed-phase LC columns compatible with formic acid enabled them to eliminate the ion suppression effects commonly observed with the reversed-phase ion paring agent trifluoroacetic acid (TFA), resulting in a one order of magnitude increase in MS sensitivity.…”

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confidence: 99%

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Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

Robinette¹,

Neamati²,

Tomer³

et al. 2006

Expert Review of Proteomics

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Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.Expert Rev. Proteomics 3(4), 399-408 (2006) In photoaffinity labeling (PAL), a ligand possessing a UV-photoactivating group is incubated with its receptor to form, first, a noncovalently bound ligand-receptor complex in which the ligand binds specifically to the binding site or pocket in the receptor due to its affinity. Some ligands, such as oligonucleotides, contain intrinsic photoactivating groups; alternatively, these groups can be introduced by synthesis into ligands for peptides and small molecules, such as drugs. It is important to establish that the incorporated photoactivating groups do not significantly alter the binding affinity of the ligand to its receptor and its functionality, compared with the nonderivatized ligand. Subsequent to the complex formation, UV irradiation triggers the photoactivating group in the ligand and leads to the conversion of the noncovalent binding into a covalent link between the ligand and its receptor at the binding site. A covalent ligand-receptor complex makes the study of the complex easier, since it permits the use of more rigorous but harsher analysis tools, such as SDS-PAGE, HPLC and mass spectrometry (MS), which would typically lead to the dissociation of the complex with information regarding the binding sites no longer being obtainable.Photoaffinity labeling with radioactively labeled ligands is a widely used method to determine the competitive binding affinities of other molecules for the binding sites in the binding pocket occupied by the radioactively labeled ligand and, as such, is a valuable tool for drug screening. When photoaffinity techniques are combined with modern MS, this approach can provide additional structural and mechanistic information regarding the protein complex, such as the ligand-receptor stoichiometry, a map of the binding pocket, and even the exact identification of the amino acid residues, which are involved in the ligand-receptor interaction. The data obtained can be used to create a 3D model of the ligand-binding pocket that might provide better insight into the nature of the ligand-receptor interaction. This knowledge can be v...

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Practical Investigation of Molecular and Biomolecular Noncovalent Recognition Processes in Solution by ESI‐MS

Schug

2009

Reactive Intermediates

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278j 8 Practical Investigation of Noncovalent Recognition by ESI-MS reduced in the gas phase. In contrast, complexes held together strictly by solvophobic forces will not survive the phase transfer process as solvent is removed. Figure 8.1 illustrates this concept. In general, it is reasonable to expect that enthalpically-driven recognition processes are most amenable to analysis by ESI-MS, whereas those that are entropically-driven must be stabilized by some enthalpic component in order to survive the electrospray process. This leads to a system dependency in the application of ESI-MS for studying noncovalent interactions, which is perhaps the most significant limitation. That said, work continues to elucidate the possibility of ESI-MS for studying hydrophobic interactions [43,44].One must also realize that ESI is a competitive ionization process. This phenomenon is best described by the work of Enke [45], and later was extended to host-guest interactions by Brodbelt and coworkers [46,47] on an equilibrium partitioning model (EPM). This model elegantly describes those factors which give rise to a limited linear dynamic range as a fixed number of charged sites on the surface of a droplet are taken up by surface-active species. This model also allows one to better understand matrix and ionization saturation effects, which can give rise to noncorrelation between gas-phase ion abundances and solution-phase concentrations as multiple analytes in the droplet compete for ionization. Species initially in the electrically neutral core of the droplet partition to the surface of the droplet according to their respective solvation energies and surface activities. The equilibrium partition model for hostguest complexation and competitive ionization is depicted in Figure 8.2. As a final general point, the preponderance of nonspecific interactions and adduct ion formation, often observed when relatively high concentrations of analytes and solution modifiers are employed, must be carefully considered, monitored, and controlled in order to avoid false positives and false negatives during both qualitative screening and quantitative binding experiments.A basic understanding of the caveats involved in performing physical binding measurements with ESI-MS is a necessity. Good experimental design is also Figure 8.

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Characterization of peptide?protein interactions using photoaffinity labeling and LC/MS

Cited by 17 publications

References 18 publications

Molecular Characterization of the Human Cα-formylglycine-generating Enzyme

Molecular Characterization of the Human Cα-formylglycine-generating Enzyme

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

Practical Investigation of Molecular and Biomolecular Noncovalent Recognition Processes in Solution by ESI‐MS

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