Alkylated Dihydroxybenzoic Acid as a MALDI Matrix Additive for Hydrophobic Peptide Analysis

Abstract

Hydrophobic peptides are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) because the majority of MALDI matrixes are hydrophilic and therefore have a low affinity for hydrophobic peptides. Here, we report on a novel matrix additive, o-alkylated dihydroxybenzoic acid (ADHB), which is a 2,5-dihydroxybenzoic acid (DHB) derivative incorporating a hydrophobic alkyl chain on a hydroxyl group to improve its affinity for hydrophobic peptides, thereby improving MALDI-MS sensitivity. The addition of ADHB to the conventional matrix α-cyano-4-hydroxycinnamic acid (CHCA) improved the sensitivity of hydrophobic peptides 10- to 100-fold. The sequence coverage of phosphorylase b digest was increased using ADHB. MS imaging indicated that hydrophobic peptides were enriched in the rim of a matrix/analyte dried spot when ADHB was used. In conclusion, the addition of ADHB to the standard matrix led to improved sensitivity of hydrophobic peptides by MALDI-MS.

MALDI Imaging Mass Spectrometry—A Mini Review of Methods and Recent Developments

“…[21][22][23][24] There are some practical parameters to keep in mind when choosing matrices. For example, the sublimation rate of your matrix in the vacuum environment of the MS instrument should be investigated; the thickness of the matrix layer should be consistent throughout the analysis, or else the signal intensity is at risk of decreasing over time.…”

(E)-Propyl α-Cyano-4-Hydroxyl Cinnamylate: A High Sensitive and Salt Tolerant Matrix for Intact Protein Profiling by MALDI Mass Spectrometry

“…Previously, 4-chloro-α-cyanocinnamic acid (Cl-CHCA), a CHCA derivative, was reported to provide lower proton affinity and obtain outstanding detection sensitivity of acidic peptides and higher sequence coverage for the identification of protein digests [29]. Recently, O-alkylated dihydroxybenzoic acid (ADHB), an esterified derivative of DHB, has been developed to improve its affinities for hydrophobic peptides [30]. ADHB as additive matrix was mixed with the traditional matrix CHCA and improved the sensitivity of hydrophobic peptides 10-to 100-fold compared with CHCA.…”

Coumarins as new matrices for matrix-assisted laser-desorption/ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of hydrophobic compounds

“…We have also developed a simple N-alkylpyridinium isotope quaternization (NAPIQ) method for improving the detection sensitivity for steroids and fatty alcohols by MALDI-MS analysis [20][21][22]. Furthermore, conventional MALDI matrices, including 2,5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxycinnamic acid (CHCA), have hydrophilic properties and low affinity for hydrophobic compounds [23,24]. Fukuyama et al have developed alkylated trihydroxyacetophenone and alkylated DHB as MALDI matrices for analysis of hydrophobic peptides [23,24].…”

“…Furthermore, conventional MALDI matrices, including 2,5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxycinnamic acid (CHCA), have hydrophilic properties and low affinity for hydrophobic compounds [23,24]. Fukuyama et al have developed alkylated trihydroxyacetophenone and alkylated DHB as MALDI matrices for analysis of hydrophobic peptides [23,24]. 6-Aza-2-thiothymine (ATT) and 4-phenyl-a-cyanocinnamic acid have been used for detecting oxidized phospholipids and lipids, respectively [25,26].…”