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

Abstract

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...

Oxidation-Induced Conformational Changes in Calcineurin Determined by Covalent Labeling and Tandem Mass Spectrometry

“…For CID-MS/MS and -MS 3 analysis of the digests from native or oxidized DMBNHS-modified CN, the mass spectrometer was operated in a DDCNL mode by performing CID-MS/MS scans on the five most intense ions from each MS survey scan, while simultaneously searching for defined neutral losses (within an m / z variance of ±0.5) corresponding to single, double, and triple S(CH 3 ) 2 neutral losses from [M N + + (m- N )H] m + precursor ions, where M represents the peptide, N represents the number of modifications, and m represents different charge states ranging from +2 to +5 (see Supplemental Table S1, Supporting Information). 31 If a predefined neutral loss was detected above a threshold abundance of 1.0 × 10 4 counts, CID-MS 3 was automatically initiated to isolate then further dissociate the most intense neutral loss product ion. For targeted ETD-MS/MS analysis of digests from native or oxidized DMBNHS-modified CN, the mass spectrometer was operated in a data-dependent mode where only predefined precursor ions of interest observed within a ± 0.5 min elution window determined from previous HPLC-ESI-MS, CID-MS/MS, and -MS 3 experiments, were selected for dissociation.…”

“…Previously, we reported the development of a strategy for mapping solvent-accessible lysine residues within proteins, via covalent labeling with a novel amine-specific protein modification reagent, S , S ′-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), and its application to a model protein, namely, human cellular retinoic acid binding protein II. 31 After reaction at different reagent to protein ratios, followed by protein digestion, capillary HPLC-ESI-MS and automated collision-induced dissociation–tandem mass spectrometry (CID-MS/MS) in an ion trap mass spectrometer, the modified peptides, and the number of modifications within each peptide, were readily identified via the dominant characteristic neutral loss(es) of a dimethylsulfide moiety from the modified lysine amino acid side chain. 31,32 The observation of the neutral losses were then used to automatically trigger the acquisition of a “data-dependent neutral loss mode” MS 3 spectrum for peptide sequence and modification site(s) characterization.…”

“…Using this approach, the experimentally determined relative accessibilities of the various lysine residues within CRABP II were found to show good agreement with those calculated from the known solution structure. 31 …”

Structural analysis of a therapeutic monoclonal antibody dimer by hydroxyl radical footprinting

“…Various labels can be utilized, including amino acid side chain-specific labeling reagents such as iodoacetamide (cysteines), N-bromosuccinimide (tryptophan), diethylpyrocarbonate (carboxyl acids), and S,S 1 -dimethylthiobutanoy lhdroxysuccinimide (lysines). [14][15][16][17] Structural information from these reagents may not be as comprehensive due to their inherent specificities.…”

Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization

“…We have previously reported that peptides containing ‘fixed charge’ dialkylsulfonium ion derivatives located on selected functional groups of certain amino acids, including the thioether side chain of methionine (formed by reaction with phenacylbromide under acidic conditions) [56-58], the thiol side chain of cysteine (formed by reaction with 3-([ N -bromoacetamido]propyl)-methylphenacylsulfonium bromide (BAPMPS)) [59], or the amino side chain of lysine or peptide N-termini (formed by reaction with S,S ′-dimethylth-iobutanoylhydroxysuccinimide ester (DMBNHS)) [60] all undergo the exclusive neutral loss(es) of dialkylsulfide groups upon CID-MS/MS, independently of the amino acid composition and precursor ion charge state (i.e. ; proton mobility), thereby enabling the selective identification of derivatized peptides from within complex mixtures.…”