Membrane Protein Analyses Using Alkylated Trihydroxyacetophenone (ATHAP) as a MALDI Matrix

Abstract

Membrane proteins containing hydrophobic regions have been difficult to analyze using MALDI-MS, probably due to the use of conventional matrices with a low affinity for hydrophobic peptides. Recently, we reported 1-(2,4,6-trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone (ATHAP)) as a matrix for hydrophobic peptides. In this study, ATHAP was applied to analyze membrane proteins containing transmembrane domains. As a result, we detected intact molecular ions for bacteriorhodopsin (BR) containing seven transmembrane domains that are difficult to detect using 2,4,6-trihydroxyacetophenone or sinapinic acid, by using ATHAP. In addition, we detected digest ions containing all seven transmembrane domains that are difficult to detect using α-cyano-4-hydroxycinnamic acid (CHCA), by using ATHAP. Moreover, ions for hydrophobic digests containing a single transmembrane domain for cadherin 1 (CDH1), fibroblast growth factor receptor 4 (FGFR4), epithelial cell adhesion molecule (EPCAM) recombinant proteins, and human epidermal growth factor receptor type 2 (HER2) were detected with higher sensitivity using ATHAP than with CHCA, confirming that ATHAP improved the membrane protein analyses, especially for hydrophobic regions such as transmembrane domains.

Advances in Ionization for Mass Spectrometry

“…These authors ascribed the success of the novel matrix vs traditional matrixes to better ablation capability and increased hydrophobicity. Similarly, Fukuyama et al also studied the ionization of hydrophobic analytes with novel matrixes . His group discovered that 1-(2,4,6-trihydroxyphenyl)­octan-1-one (alkylated trihydroxyacetophenone (ATHAP)) is an excellent matrix for the analysis of hydrophobic membrane proteins containing transmembrane domains.…”

Inverse Hydrogen-Bonding Change Between the Protonated Retinal Schiff Base and Water Molecules upon Photoisomerization in Heliorhodopsin 48C12

“…Additionally, 0.1 μg/μL trypsin at an enzyme/substrate ratio of 1:5 (w/w) was added and the obtained mixtures were then incubated at 37 °C overnight. The obtained tryptic digestion solution was mixed with 10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA, in 0.05% trifluoroacetic acid with 50% acetonitrile) at a digestion solution/CHCA ratio of 1:5 (v/v) . The digestion of HeR 48C12 into 17 peptide fragments is expected, as shown in Table S2.…”

Advances in mass spectrometry imaging for spatial cancer metabolomics

“…Positive/negative Lipids (Garate et al, 2015;Perry et al, 2020;Thomas et al, 2012) 2,5-Dihydroxyacetophenone (2,5-DHA) Positive/negative Lipids (Bien et al, 2020;Claes et al, 2021) Proteins (Kaya et al, 2020;Zavalin et al, 2015) 2,6-Dihydroxyacetophenone (2,6-DHA) Negative Lipids (Jackson et al, 2018) 3-Hydroxypicolinic acid (3-HPA) Negative Oligonucleotides (Yokoi et al, 2018) Lipids (Thomas et al, 2012) Caffeic acid (CA) Positive Proteins (H. Liu et al, 2021) 2′,4′,6′-Trihydroxyacetophenone (THAP) Positive/negative Lipids (Basu et al, 2019) Proteins (alkylated THAP used) (Fukuyama et al, 2016) 1,8-Bis (dimethylamino) naphthalene (DMAN) Negative Metabolites (Ye et al, 2013) 1,8-Di (piperidinyl)-naphthalene (DPN) Negative Lipids (Weißflog & Svatoš, 2016) 1,8,9-Anthracentriol (DIT) Positive/negative Lipids (Thomas et al, 2012) Quercetin Positive/negative Lipids (X. Wang et al, 2014)…”