Alkylated Trihydroxyacetophenone as a MALDI Matrix for Hydrophobic Peptides

Hydrophobic peptides are difficult to detect in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because of the hydrophilic properties of conventional matrices and the low affinity for hydrophobic peptides. Recently, we reported on alkylated dihydroxybenzoic acid (ADHB) as a matrix additive for hydrophobic peptides; however, the peptides were detected in the rim of the matrix-analyte dried spot. Here, we report on a novel matrix, alkylated trihydroxyacetophenone (ATHAP), which is a 2,4,6-trihydroxyacetophenone derivative incorporating a hydrophobic alkyl chain on the acetyl group and thus is expected to have an affinity for hydrophobic peptides. ATHAP increased the sensitivity of hydrophobic peptides 10-fold compared with α-cyano-4-hydroxycinnamic acid (CHCA), in which the detection of hydrophilic peptides was suppressed. The peptides were detected throughout the entire matrix-analyte dried spot using ATHAP, overcoming the difficulty of finding a “sweet spot” when using ADHB. In addition, ATHAP functioned alone as a matrix, unlike ADHB as an additive. In phosphorylase b digests analysis, hydrophobic peptides, which were not detected with CHCA for 1 pmol, were detected with this matrix, confirming that ATHAP led to increased sequence coverage and may extend the range of target analytes in MALDI-MS

Imidazole C‑2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine C^ε1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second-order with respect to the concentrations of the imidazolium cation and OD^– in D₂O. The second-order rate constant (<i>k</i>₂) of this reaction was calculated from the pH dependency of the pseudo-first-order rate constant (<i>k</i>_φ) obtained from the change in average mass [Δ<i>M</i>_r (0 ≤ Δ<i>M</i>_r < 1)] of a peptide fragment containing a defined histidine residue at incubation time (<i>t</i>) such that <i>k</i>_φ = −[ln­(1 – Δ<i>M</i>_r)]/<i>t</i>. We preferred using <i>k</i>₂ rather than <i>k</i>_φ because <i>k</i>₂^max (maximal value of <i>k</i>₂) was empirically related to p<i>K</i>_a as illustrated with a Brønsted plot [log <i>k</i>₂^max = −0.7p<i>K</i>_a + α (α is an arbitrary constant)], so that we could analyze the effect of structure on the H/D exchange rate in terms of log­(<i>k</i>₂^max/<i>k</i>₂) representing the deviation of <i>k</i>₂ from <i>k</i>₂^max. In the catalytic site of bovine ribonuclease A, His12 showed a change in log­(<i>k</i>₂^max/<i>k</i>₂) much larger than that of His119 upon binding with cytidine 3′-monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, we need to consider the hydrogen bonds of the imidazole group with nondissociable groups to interpret an extremely slow H/D exchange rate of His48 in a partially solvent-exposed situation