Molecular Characterization of Surface Topology in Protein Tertiary Structures by Amino-Acylation and Mass Spectrometric Peptide Mapping

Glocker, Michael O.; Borchers, Christoph H.; Fiedler, W.; Suckau, Detlev; Przybylski, Michael

doi:10.1021/bc00030a014

Cited by 135 publications

(143 citation statements)

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“…Recent advances in mass spectrometry have made it possible to assess the reactivities of amino acid side chains without the use of radioactive materials (Qin et al, 1992;Akashi et al, 1993;Hasan et al, 1993;Glocker et al, 1994). Due to the high specificity of mass spectrometry, modified peptides can be detected and identified even when isolation is incomplete.…”

Section: Advances In Experimental Methodsmentioning

confidence: 99%

The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA

1998

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Differential chemical modification of the lysines and amino-terminus of Escherichia coli single-strand binding (SSB) protein was used to determine their roles in the binding of SSB to single-stranded DNA (ssDNA). A combination of isotope labeling and mass spectrometry was used to determine the rates at which SSB was acetylated by acetic anhydride. First, SSB was labeled by deuterated acetic anhydride for given lengths of time in the presence or absence of single-stranded ssDNA. Then, the protein was denatured and completely acetylated by nondeuterated acetic anhydride. Enzymatic digests of the completely acetylated, isotopically labeled SSB were analyzed by electrospray ionization mass spectrometry. The intensities of the deuterated and nondeuterated forms of acetylated peptides provided accurate quantification of the reactivity of the amines in native SSB, either free or bound to ssDNA. Acetylation rate constants were determined from time course measurements. In the absence of ssDNA, the terminal a-amine of SSB was IO-fold more reactive than Lys residues at positions 43, 62, 73, and 87. The reactivities of Lys 7 and 49 were much lower yet, suggesting that they have very limited access to solution under any condition. In the presence of ssDNA, the reactivities of the amino-terminus and Lys residues 43, 62, 73, and 87 were reduced by factors of 3.7-25, indicating that the environments around all of these amines is substantially altered by binding of SSB to ssDNA. Three of these residues are located near putative ssDNA binding sites, whereas Lys 87 is located at the monomer-monomer interface.

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Section: Advances In Experimental Methodsmentioning

confidence: 99%

The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA

1998

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“…The reaction was carried out as described previously [24]. Insulin was dissolved in 8 M urea in 0.1 M NH 4 HCO 3 with an approximate protein concentration of 400 M. A freshly prepared solution of DTT was added in a 50-fold molar excess with regard to cysteine.…”

Section: Cleavage Of Disulfide Bonds and Carbamidation Of Free Cysteinementioning

confidence: 99%

Investigation of the influence of charge derivatization on the fragmentation of multiply protonated peptides

Sonsmann¹,

Römer²,

Schomburg³

2002

J. Am. Soc. Mass Spectrom.

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The fragmentation of the multiply charged peptides b-chain of bovine insulin and glucagon have been investigated under low energy collision induced dissociation (CID) conditions using an electrospray ion trap mass spectrometer. The influence of charge state, specific amino acids such as aspartate or proline, the location of basic sites, and the derivatization on the fragmentation behavior has been the focus of interest. As a basis for understanding the fragmentation process, the concept of the mobile proton was applied. A set of different derivatives was used to manipulate the sites of protonation of the peptides in order to control and improve the fragmentation behavior. These results can be applied for de novo sequencing, although the sequence-specific fragmentation processes have significant influence on the dissociation behavior of the peptides. (J Am Soc Mass Spectrom 2002, 13, 47-58)

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“…A conformational change involving unfolding of the 127-165-amino acid loop would expose residues Lys 185 and Lys 220 , which would then be able to be cross-linked. This hypothesis is currently being tested in our laboratory by other structural proteomics methods, such as limited proteolysis (58), surface modification (59), and hydrogen/deuterium exchange (60,61). …”

Section: Discussionmentioning

confidence: 99%

Use of Proteinase K Nonspecific Digestion for Selective and Comprehensive Identification of Interpeptide Cross-links: Application to Prion Proteins

Petrotchenko

Serpa

Hardie

et al. 2012

Molecular & Cellular Proteomics

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Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight crosslinked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP C ) and oligomeric form of prion protein (PrP␤). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the crosslinked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP Structural proteomics, which combines protein chemistry methods with modern mass spectrometry techniques for protein and peptides, is an emerging technology in structural biology. One of the tools for modern structural proteomics is chemical cross-linking combined with mass spectrometry (1-5). Cross-linking analysis provides the distance between two cross-linked amino acid residues, whereas mass spectrometry provides information on which residues are connected. The basis of this method is a chemical reaction of the crosslinking reagent with functional groups on a protein, leading to the formation of a covalent bond between each end of the reagent and the protein. The distance between two crosslinked sites is determined by the length of the spacer in the cross-linking reagent. Thus, identification of the cross-linked sites on a protein or a protein complex provides spatial information and distance constraints for the two amino acid residues that are connected by the cross-linker.The use of chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics (4, 6 -8). In recent years, we and others have been developing an array of cross-linking and modification reagents, software, and methods specifically designed for protein cross-linking experiments combined with MS analysis (9 -18). For mass spectrometric determina...

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Molecular Characterization of Surface Topology in Protein Tertiary Structures by Amino-Acylation and Mass Spectrometric Peptide Mapping

Cited by 135 publications

References 41 publications

The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA

The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA

Investigation of the influence of charge derivatization on the fragmentation of multiply protonated peptides

Use of Proteinase K Nonspecific Digestion for Selective and Comprehensive Identification of Interpeptide Cross-links: Application to Prion Proteins

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