Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

Scholten, Arjen; Visser, N.F.C.; Heuvel, Robert H. H. van den; Heck, Albert J. R.

doi:10.1016/j.jasms.2006.03.005

Cited by 31 publications

(31 citation statements)

References 39 publications

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“…For example, unmodified y 3 , y 4 , y 5 , and y 6 product ions along with modified y 7 and y 8 ions indicate that His13 is the modified residue in the Ser11-Lys19 fragment of β2m ( Figure 1a). Similarly, unmodified b ions from b 8 through b 14 , a modified b 15 ion, and a complete series of modified y ions from y 2 to y 13 indicate that Ser117 is the modified residue in the Tyr103-Lys119 fragment of myoglobin ( Figure 1b). The MS/MS spectra for the remaining 15 modified peptides are included in the Supporting Information, and these spectra confirm the modification sites that are reported in Tables 1 and 2. DEPC is known to react with Tyr residues in addition to His residues, but the modification of Ser and Thr residues is previously unreported as far as we are aware.…”

Section: Resultsmentioning

confidence: 96%

“…[12][13][14][15][16][17][18][19][20] Because protein conformational changes affect their surface topology, amino acid residues involved in the structural changes can be identified from differential modification patterns. Information from surface mapping experiments is reliable, though, only if the structural integrity of a protein is preserved during the reaction.…”

Section: Introductionmentioning

confidence: 99%

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Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

2008

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The reliability and information content of diethylpyrocarbonate (DEPC) as a covalent probe of protein surface structure has been improved when used appropriately with mass spectrometric detection. Using myoglobin, cytochrome c, and β-2-microglobulin as model protein systems, we demonstrate for the first time that DEPC can modify Ser and Thr residues in addition to His and Tyr residues. This result expands the capability of DEPC as a structural probe because about 25% of the sequence of the average protein can now be covered using this covalent labeling reagent. In addition, we establish a new approach based on mass spectrometry to ensure the structural integrity of proteins during amino acid-specific covalent labeling reactions. This approach involves monitoring the extent of modification as a function of reagent concentration and allows any small-scale or local perturbations caused by the covalent label to be readily identified and avoided. Results indicate that these dose-response plots are much more reliable and generally applicable probes of possible protein structural changes than fluorescence or circular dichroism spectroscopies. These dose-response plots also provide a means of quantitatively comparing the reactivity of each modified residue. Based on comparisons to known X-ray crystal structures, we find that the solvent accessibility of the reactive atom in the side chain and the presence of a nearby charged residue most affect modification rates. Finally, this improved surface mapping method has been used to determine the effect of Cu(II) binding on the structure of β-2-microglobulin. Results confirm that Cu(II) binds His31, but not any of the other three His residues, and changes the solvent accessibility of residues near His31 and near the N-terminus.

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Section: Resultsmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

2008

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“…Acetic anhydride was the earliest lysine derivatization reagent used [18]. More recently, however, lysine targeted labeling strategies have shifted towards the use of N-hydroxysuccinimide (NHS) esters, due to their improved reaction specificity and a roughly 1000-fold lower required concentration compared with acetic anhydride [22][23][24][25][26]. Lysine-specific modification by S-methylthioacetimidate has also been recently reported [27].…”

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confidence: 99%

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

Zhou

Wang

et al. 2010

J. Am. Soc. Mass Spectrom.

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Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. However, a common problem associated with the use of selective chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the 'needle in a haystack' problem). To address this challenge, we describe here the development of a strategy involving the synthesis and application of a novel 'fixed charge' sulfonium ion containing lysine-specific protein modification reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-ESI-MS, automated CID-MS/MS, and data-dependant neutral loss mode MS 3 in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, cellular retinoic acid binding protein II (CRABP II). After reaction with different reagent:protein ratios and digestion with Glu-C, modified peptides are selectively identified and the number of modifications within each peptide are determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility) of the peptide. The observation of these characteristic neutral losses are then used to automatically 'trigger' the acquisition of an MS 3 spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II. (J Am Soc Mass Spectrom 2010, 21, 1339 -1351) © 2010 American Society for Mass Spectrometry M ass spectrometry (MS) combined with protein labeling has found increasing utility as an alternative tool to conventional high-resolution methods such as X-ray crystallography or NMR for the examination of higher order protein structure (e.g., tertiary and quaternary), conformation, ligand binding, and dynamics [1]. Although typically providing lower resolution than these more established approaches, MS-based analysis strategies have particular advantages in sensitivity and speed, and the capability of analyzing proteins or protein complexes that are not amenable to crystallization, or that fall outside the mass range routinely accessible by NMR.A variety of MS/protein labeling methods have been developed, and are based on either the use of (1) hydrogen-deuterium exchange (HDX) [2,3] or (2) stable chemical modification [4 -6], before proteolytic digestion and analysis by HPLC-MS and/or tandem mass spectrometry (MS/MS). HDX may potentially be used to probe the entire protein backbone, thereby providing the highest resolution of the MS-based approaches. However, limitations of HDX that can hamper determination...

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“…One of the most utilized chemical labeling methods is lysine residue modification [2][3][4][5][6]. Lysine residue labeling offers many advantages for studying protein solvent accessibility, one of which is the unique reactivity of the lysine side-chain primary amine.…”

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confidence: 99%

Solution insights into the structure of the Efb/C3 complement inhibitory complex as revealed by lysine acetylation and mass spectrometry

Chen

Schuster

Sfyroera

et al. 2008

J. Am. Soc. Mass Spectrom.

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The extracellular fibrinogen-binding protein (Efb), an immunosuppressive and anti-inflammatory protein secreted by Staphylococcus aureus, has been identified as a potent inhibitor of complement-mediated innate immunity. Efb functions by binding to and disrupting the function of complement component 3 (C3). In a recent study, we presented a high-resolution co-crystal structure of the complement inhibitory domain of Efb (Efb-C) bound to its cognate domain (C3d) from human C3 and employed a series of structure/function analyses that provided evidence for an entirely new, conformational change-based mechanism of complement inhibition. To better understand the Efb/C3 complex and its downstream effects on C3 inhibition, we investigated the solvent-accessibility and protein interface of Efb(-C)/C3d using a method of lysine acetylation, proteolytic digestion, and mass spectrometric analysis. Lysine modification in Efb was monitored by the mass increment of lysine-containing fragments. Besides confirming the binding sites observed in co-crystal structure study, the in-solution data presented here suggest additional contacting point(s) between the proteins that were not revealed by crystallography. The results of this study demonstrate that solution-based analysis of protein-protein interactions can provide important complementary information on the nature of protein-protein interactions. (J Am Soc Mass Spectrom 2008, 19, 55-65)

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Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

Cited by 31 publications

References 39 publications

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

Solution insights into the structure of the Efb/C3 complement inhibitory complex as revealed by lysine acetylation and mass spectrometry

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