We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS 3 ). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the -trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1, to build an FGF-2 homology model with a backbone error of 4.8 Å (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation. In recent years, the number of novel proteins identified by genomic (1, 2) and proteomic projects has dramatically increased, with a concomitant need for more rapid determination of their tertiary structures.Visualization of the three-dimensional structures of proteins has traditionally been realized by x-ray crystallography and NMR. These techniques produce high resolution atomic data but require relatively large amounts (10 to 100 mg) of pure analyte in a particular solution or crystalline state. Even if these conditions are met, it can take months or even years to generate a molecular structure by following these methodologies.To develop an alternative approach to structure determination that could keep pace with the rate of novel protein identification, we have re-examined cross-linking technology in the light of newer analytical protocols for the separation and identification of complex peptide mixtures. Previous investigators have shown that cross-linking experiments can provide low resolution interatomic distance information (3). In theory, given enough distance information, it is possible to solve the tertiary structure of a macromolecule (4, 5).The challenge we faced in trying to generate such information in a short time using cross-linking technology was to devise a rapid method for identifying cross-linked residues. MS affords high throughput but has rarely been used for the identification of cross-links. One study has been published where disuccinimidyl ester cross-linking, Edman sequencing, and MS were used to validate a model of human erythropoietin (6). Recent advances in MS (7, 8) gave us the means whereby we could determine the masses and sequences of large peptides with high accuracy and sensitivity (9, 10). These improvements make it feasible to analyze complex peptide mixtures from proteolytically digested, cross-linked proteins (11) very quickly. Specifically, we describe the use of chemical cross-linking and time-of-flight (TOF) MS to identify Lys-Lys cross-links. We also show how t...
In a previous report (Young et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802-5806), we provided a proof-of-principle for fold recognition of proteins using a homobifunctional amine-specific chemical crosslinking reagent in combination with mass spectrometry analysis and homology modeling. In this current work, we propose a systematic nomenclature to describe the types of peptides that are generated after proteolysis of crosslinked proteins, their fragmentation by tandem mass spectrometry, and an automated algorithm for MS/MS spectral assignment called "MS2Assign." Several examples are provided from crosslinked peptides and proteins including HIV-integrase, cytochrome c, ribonuclease A, myoglobin, cytidine 5-monophosphate N-acetylneuraminic acid synthetase, and the peptide thymopentin. Tandem mass spectra were obtained from various crosslinked peptides using post source decay MALDI-TOF and collision induced dissociation on a quadrupole-TOF instrument, along with their automated interpretation using MS2Assign. A variety of possible outcomes are described and categorized according to the number of modified lysines and/or peptide chains involved, as well as the presence of singly modified (dead-end) lysine residues. In addition, the proteolysis and chromatographic conditions necessary for optimized crosslinked peptide recovery are presented.
Mass spectrometric analysis of wild-type proteins that have been covalently modified by bifunctional cross-linking reagents and then digested proteolytically can be used to obtain low-resolution distance constraints, which can be useful for protein structure determination. Limitations of this approach include time-consuming separation steps, such as the separation of internally cross-linked protein monomers from covalent dimers, and a susceptibility to artifacts due to low levels of natural and man-made peptide modifications that can be mistaken for cross-linked species. The results presented here show that when a crude cross-linked protein mixture is injected into an electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) instrument, the cross-link positions can be localized by fragmentation and mass spectrometry on the 'gas-phase purified' singly internally cross-linked monomer. Our results show that reaction of ubiquitin with the homobifunctional lysine-lysine cross-linking reagent dissuccinimidyl suberate (DSS) resulted in two cross-links consistent with the known ubiquitin tertiary structure (K6-K11 and K48-K63). Because no protein or peptide chemistry steps are needed, other than the initial cross-linking, this new top down approach appears well suited for high-throughput experiments with multiple cross-linkers and reaction conditions. Published in 2002 by John Wiley& Sons, Ltd.
A top-down approach based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and beta-ethoxy-alpha-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chemistry to produce strand cleavage at the modified nucleotides, and analysis by polyacrylamide gel electrophoresis to determine the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the analysis of individual target adducts present in complex reaction mixtures with no need for separation techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MS(n)() experiments to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated reduction and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data reduction module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calculates the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data reduction program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on cross-linking strategies and top-down ESI-FTMS.
We present a method employing top-down Fourier transform mass spectrometry (FTMS) for the rapid profiling of amino acid side-chain reactivity. The reactivity of side-chain groups can be used to infer residue-specific solvent accessibility and can also be used in the same way as H/D exchange reactions to probe protein structure and interactions. We probed the reactivity of the N-terminal and epsilon-lysine amino groups of ubiquitin by reaction with N-hydroxysuccinimidyl acetate (NHSAc), which specifically acetylates primary amines. Using a hybrid Q-FTMS instrument, we observed several series of multiply acetylated ubiquitin ions that varied with the NHSAc:protein stoichiometry. We isolated and fragmented each member of the series of acetylated ubiquitin ions in the front end of the instrument and measured the fragment ion masses in the FTMS analyzer cell to determine which residue positions were modified. As we increased the NHSAc:protein stoichiometric ratio, 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 (Met 1 approximately Lys 6 approximately Lys 48 approximately Lys 63>Lys 33>Lys 11>Lys 27, Lys 29). These results are in excellent agreement with the reactivity expected from other studies and predicted from the known crystal structure of ubiquitin. The top-down approach eliminates the need for proteolytic digestion, high-performance liquid chromatographic separations and all other chemical steps except the labeling reaction, making it rapid and amenable to automation using small quantities of protein.
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