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

Abstract

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

Identification of cross-linked peptides from large sequence databases

“…To exploit this information, both peptides that are involved in a crosslink need to be identified. Improvement in mass spectrometry (MS) technology has made this conceivable, in particular high mass precision spectrometers and the development of isotopically coded cross-linkers [3][4][5] . Several studies have proven the general feasibility of this approach 3,[6][7][8][9] .…”

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

“…These include incorporating an affinity tag into the linker for peptide purification [7][8][9][10][11][12][13][14], as well as various isotope labeling strategies [15][16][17][18][19]. These methods, however, often yield complex, difficult to analyze MS/MS data, as the intermolecular cross-links consist of two peptides covalently joined, and product ions from both peptides are present in the spectrum [20].…”

Development of Large-scale Cross-linking Mass Spectrometry

“…For example 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) results in the presence of a 66Da doublet peak and disappearance of the crosslinked peak after addition of reducing agent (17,(19)(20)(21)(22)(23). But only Protein Prospector software considers reporter ions that are naturally embedded in every cross-linked lysine regardless which cross-linker is used.…”