Partial Acetylation of Lysine Residues Improves Intraprotein Cross-Linking

Abstract

Intramolecular cross-linking coupled with mass spectrometric identification of cross-linked amino acids is a rapid method for elucidating low-resolution protein tertiary structures or fold families. However, previous cross-linking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide cross-links that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein cross-linking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyl-d3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with acetic-d6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial N-acetylation of the most reactive lysine residues prior to cross-linking can redirect and increase the observable Lys-Lys cross-links, partially acetylated bovine cytochrome c was cross-linked with the amine-specific, bis-functional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing Lys-Lys cross-links, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional cross-linked peptides contained novel Lys-Lys cross-link information not seen in the non-acetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold.

Traveling-wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking

“…From this observation, one can infer that for P2, a higher pH condition favors modification on the first Lys residue (unmodified y 6 ions of m/z 676.3), slightly increasing its proportion with respect to modification on the second Lys residue, which presents a higher reactivity at lower pH. Differences in reactivity of individual Lys residues have also been observed in a study performed by Guo et al [69], in which bovine cytochrome c was subjected to chemical cross-linking after partial acetylation of its Lys residues. It was shown that the pKa of Lys residues in the protein varies over two units, which can be correlated to differences in their chemical environment, steric hindrance effects, and solvent accessibility.…”

“…The use of NHS esters for cross-linking strategies has been applied for more than 30 y to identify binding partners, [11] and a more recent approach relies on protein cross-linking coupled to mass spectrometry (MS) experiments to map three-dimensional structures of proteins (MS3D) [12,13]. Instruction manuals from commercial NHS-ester cross-linkers and most application literature state that NHS esters react exclusively with primary amines (N-terminus and -amino group of Lys side chains), and that when reacted with sulfhydryl or hydroxyl groups, do not yield stable products [14,15]. However, recent studies showed that the side chains of other residues (such as Tyr and Ser) can also sometimes yield stable cross-link species when reacted with NHS esters [16 -18].…”

Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

“…First, recombinant His-tagged mouse LCAD (hereafter called LCAD) was incubated with the chemical acetylating agent acetic anhydride at a ratio of 1:1 anhydride to lysine, as calculated on the basis of the known 27 lysine residues per LCAD monomer (34). LCAD became highly acetylated, as detected by Western blotting with anti-acetyllysine antibodies (Fig.…”

“…We therefore tested the effects of sulfo-NHS-acetate on LCAD. Sulfo-NHS-acetate is commonly used to acetylate and block surface lysines for proteomics studies and is known to be more stable than acetic anhydride under physiologically relevant conditions (34). Aliquots of LCAD were acetylated with increasing concentrations of sulfo-NHS-acetate and used as a substrate for SIRT3 deacetylation assays.…”

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

“…The M1 amine is particularly reactive because it has a lower pKa-value than lysine amine groups. That the different amines/lysine residues in proteins show different reactivity with amine-specific crosslinkers has previously been recognized, and acetylation of amine groups prior to crosslinking has been proposed to avoid exclusive identification of crosslinks involving the most reactive lysine residues only (Guo et al 2008). Furthermore, the M1 amine is also located at the very end of a flexible N-terminal arm in a a The observation(s) of the same peptide pair but with one or two methionine residues oxidized strengthens the identification, and in this case, the MS spectrum of the oxidized version of the crosslink is also shown in Fig In presence of MDH, the number of crosslinks M1 makes to Hsp21 itself is reduced compared with the situation where no MDH is present (Lambert et al 2011b;Söderberg et al 2012), and instead, the Hsp21 M1 amine crosslinks to MDH (Table 1).…”