A Top‐down method for the determination of residue‐specific solvent accessibility in proteins

Novák, Petr; Kruppa, Gary; Young, Malin M.; Schoeniger, Joe

doi:10.1002/jms.587

Cited by 66 publications

(85 citation statements)

References 27 publications

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“…Previous studies have shown that reactivity is influenced mainly by the solvent accessibility of the amino acid and of the reactive atom. 23,25,29,30 The rate coefficients in Tables 1 and 2 indicate that there is not a simple correlation between the modification rates and the solvent accessibility of the entire side chain. The % SASA ratio does not seem to always correlate with the reaction rates.…”

Section: Factors Affecting the Reactivity Of Functional Groupsmentioning

confidence: 99%

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: Factors Affecting the Reactivity Of Functional Groupsmentioning

confidence: 99%

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

2008

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“…This instrument was described in detail in a previous publication [11]. After labeling or cross-linking and purification, protein samples were diluted to 10 M in 50:50 MeOH:H 2 O with 6% acetic acid and were introduced via direct infusion to an Apollo ESI ion source (Bruker Daltonics, Billerica, MA) at a flow rate of 1.5 L/min.…”

Section: Sample Analysismentioning

confidence: 99%

“…Examination of the known structure of ubiquitin showed that there are many geometrically allowed cross-links be-tween primary amino groups that were not observed experimentally. To determine whether our method could not localize these cross-links or if the cross-links were not formed, we probed the reactivity of the primary amino groups of ubiquitin by reaction with N-hydroxysuccinimidyl acetate (NHSAc), which specifically acetylates primary amines by the elimination of N-hydroxy succinimide; this is the same elimination reaction that occurs twice in the cross-linking reaction with the disuccinimidyl esters [11]. As the stoichiometric ratio of NHSAc:protein increased, identification of the fragments from native protein and protein with successively increasing modification allowed the assignment of the complete order of reactivity of the primary amino groups in ubiquitin (Met1 Ϸ Lys 6 Ϸ Lys 48 Ϸ Lys 63 Ͼ Lys 33 Ͼ Lys11 Ͼ Lys 27, Lys 29).…”

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confidence: 99%

Strategy for selective chemical cross-linking of tyrosine and lysine residues

Leavell

Novák

Behrens

et al. 2004

J. Am. Soc. Mass Spectrom.

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Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin ␤-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminustyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH. I nter-molecular chemical cross-linking has a long history as a tool for the study of the quaternary structure of protein complexes [1,2], and many years ago it was suggested that intra-molecular cross-linking could be used as a method of obtaining distance constraints that would be useful in developing structural models of proteins [3]. However, the promise of intra-molecular crosslinking for structural modeling has only recently been enabled by state of the art mass spectrometric methods that make determination of the cross-linked residues in a protein practical on a reasonable time scale and with small quantities of protein. The first study to derive a sufficient number of intra-molecular distance constraints to develop a structural model for a protein used fibroblast growth factor 2 (FGF-2) as a test case [4]. FGF-2 is a 17 kDa protein, which has 14 lysines evenly dispersed in its 155 residue sequence. Using only the commercially available primary amine reactive cross-linker bis(sulfosuccinimidyl) suberate, 18 cross-links were found in FGF-2 in this study, of which 15 provided useful distance constraints for determining the fold family of the protein. Using these 15 constraints it was possible to assign the FGF2 to the corr...

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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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A Top‐down method for the determination of residue‐specific solvent accessibility in proteins

Cited by 66 publications

References 27 publications

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

Strategy for selective chemical cross-linking of tyrosine and lysine residues

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

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