A top down approach to protein structural studies using chemical cross‐linking and Fourier transform mass spectrometry

For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d 0 and d 4 ) cross-linkers BS 3 (bis[sulfosuccinimidyl]suberate) and BS 2 G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central ␣-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few

mentioning

confidence: 99%

Isotope-labeled cross-linkers and fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex

Ihling

Schmidt

Kalkhof

et al. 2006

“…In our own recent work, we showed that a top down approach to the localization of cross-links using FTMS could be applied to localize three cross-links in ubiquitin [7], and in a more recent publication we showed that the distance constraints can be improved by using a series of cross-linkers of different lengths [6]. However, ubiquitin has eight primary amino groups (seven on lysines and one at the amino terminus) that should be reactive with dissuccinimidyl esters, the reagents that were used in the preliminary studies described above, yet only three cross-links were observed.…”

mentioning

confidence: 99%

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

Leavell

Novák

Behrens

et al. 2004

Self Cite

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...

“…Cross-linkages may be either intramolecular or intermolecular. Intramolecular cross-linking demonstrates which regions within a protein are adjacent [1][2][3], while intermolecular cross-linking indicates adjacent regions of subunits in a protein assembly [4 -6]. To identify cross-linked residues, proteins are digested into peptides whose identities are determined by mass spectrometry (MS).…”

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confidence: 99%

Unexpected products from the reaction of the synthetic cross-linker 3,3′-dithiobis(sulfosuccinimidyl propionate), DTSSP with peptides

Swaim

Smith

2004

Synthetic cross-linking reagents, such as 3,3Ј-dithiobis(sulfosuccinimidyl propionate), DTSSP, can react with sidechains of amino acids that are within close proximity. Identification of cross-linked residues provides insight into the folded structures of proteins. However, analysis of proteolytic digests of proteins cross-linked with commercially available DTSSP is difficult because many ions cannot be attributed to reported reactions of DTSSP. To better understand the reactivity of DTSSP, products from the reaction of DTSSP with several model peptides were analyzed by HPLC electrospray ionization mass spectrometry (ESIMS). Several products not previously reported were identified. Sources for these unexpected products were traced to reaction of DTSSP with contaminant ammonium ions in the buffer, to reaction of contaminants present in the commercial DTSSP reagent, and to reactivity of DTSSP with serine and tyrosine residues. In addition, the collision-induced-dissociation (CID) of peptides modified by DTSSP was investigated. These results showed that certain DTSSP-peptide adducts easily undergo in-source fragmentation to give additional unexpected ions. This study of the reactions of DTSSP with model peptides has revealed the major types of ions that are likely to be found in proteolytic digests of proteins cross-linked with DTSSP, thereby facilitating identification of the cross-linked residues that can provide information about the three-dimensional structures of folded proteins. (J Am Soc Mass Spectrom 2004, 15, 736 -749)