Probing Protein Tertiary Structure with Amidination

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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confidence: 99%

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

Zhou

Wang

et al. 2010

“…One of the most utilized chemical labeling methods is lysine residue modification [2][3][4][5][6]. Lysine residue labeling offers many advantages for studying protein solvent accessibility, one of which is the unique reactivity of the lysine side-chain primary amine.…”

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confidence: 99%

Solution insights into the structure of the Efb/C3 complement inhibitory complex as revealed by lysine acetylation and mass spectrometry

Chen

Schuster

Sfyroera

et al. 2008

The extracellular fibrinogen-binding protein (Efb), an immunosuppressive and anti-inflammatory protein secreted by Staphylococcus aureus, has been identified as a potent inhibitor of complement-mediated innate immunity. Efb functions by binding to and disrupting the function of complement component 3 (C3). In a recent study, we presented a high-resolution co-crystal structure of the complement inhibitory domain of Efb (Efb-C) bound to its cognate domain (C3d) from human C3 and employed a series of structure/function analyses that provided evidence for an entirely new, conformational change-based mechanism of complement inhibition. To better understand the Efb/C3 complex and its downstream effects on C3 inhibition, we investigated the solvent-accessibility and protein interface of Efb(-C)/C3d using a method of lysine acetylation, proteolytic digestion, and mass spectrometric analysis. Lysine modification in Efb was monitored by the mass increment of lysine-containing fragments. Besides confirming the binding sites observed in co-crystal structure study, the in-solution data presented here suggest additional contacting point(s) between the proteins that were not revealed by crystallography. The results of this study demonstrate that solution-based analysis of protein-protein interactions can provide important complementary information on the nature of protein-protein interactions. (J Am Soc Mass Spectrom 2008, 19, 55-65)

“…Others have also successfully used acetic anhydride for structure determinations [9,10]. Recently, several new reagents and labels have emerged that target solvent accessible lysines: N-hydroxysuccinimido-biotin (NHS-biotin) [11,12], sulfosuccinimidyl acetate [13] and S-methylthioacetimidate [14]. Especially, the use of NHS-ester chemistry in lysine labeling has proven to be efficient and reliable.…”

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confidence: 99%

Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

Scholten

Visser

Heuvel

et al. 2006

To understand how proteins perform their function, knowledge about their structure and dynamics is essential. Here we use a combination of an efficient chemical lysine acetylation reaction and nanoLC-MALDI tandem mass spectrometry to probe the accessibility of every lysine residue in a protein complex. To demonstrate the applicability of this approach, we studied the interaction between the DNase domain of Colicin E9 (E9) and its immunity protein Im9. Free E9 and E9 in complex with Im9 were rapidly acetylated, followed by proteolytic digestion and analysis by LC-MALDI-TOF/TOF MS/MS. Acetylated peptides could be filtered out of the complex peptide mixtures using selective ion chromatograms of the specific immonium marker ions. Additionally, isobaric acetylated peptides, acetylated at different sites, could be separated by their LC retention times. The combination of LC and MALDI-TOF/TOF MS/MS provided information about the amount of acetylation on each individual lysine even for peptides containing several lysine residues. In general, our data agree well with those derived from the crystal structure of E9 and the E9:Im9 complex. Interestingly, next to in the binding interface expected lysines, K89 and K97, two from the crystal structure data unexpected lysines, K81 and K76, were observed to become less exposed upon Im9 binding. Moreover, K55 and K63, positioned in the predicted DNA binding region, were also found to be less accessible upon Im9 binding. These findings may illustrate some of the described differences in the solution-phase structure of the E9:Im9 complex compared with the crystal structure. (J Am Soc Mass Spectrom 2006, 17, 983-994)