Use of 2,6-Dihydroxyacetophenone for Analysis of Fragile Peptides, Disulphide Bonding and Small Proteins by Matrix-assisted Laser Desorption/Ionization

Abstract

Several peptides were shown to undergo fragmentation during matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to a degree which complicated their analysis using alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix, even at threshold laser irradiance. These peptides included synthetic peptides, peptides isolated from viral proteins and a phosphopeptide from beta-casein (residues 33-48). The excessive fragmentation occurred usually as a post-source phenomenon; however, in-source fragmentation was also observed. The combined effects of in-source and post-source fragmentation of one peptide studied led to a failure to observe the protonated molecule of this peptide in reflector mode analysis. The phosphopeptide studied exhibited a high degree of beta-elimination of phosphate. It was demonstrated that the fragility exhibited by these peptides in CHCA, including beta-elimination of phosphate from serine, was not evident with a matrix comprising 2,6-dihydroxyacetophenone (DHAP) and di-ammonium hydrogen citrate (DAHC). The DHAP/DAHC matrix was also adapted for direct analysis of peptides from an acidic reducing milieu containing tris(2-carboxyethyl)phosphine. The molecular weight of equine cytochrome c was determined with a relatively high degree of accuracy (experimental M(r) = 12360.2 +/- 1.4 Da compared to the theoretical M(r) = 12360.09 Da) using DHAP/DAHC as a matrix for reflector mode analysis.

Synthesis and characterization of histidine‐phosphorylated peptides

“…However, no h t H + was detected for the phosphohistidine-containing peptides in positive-ion MALDI analysis, when CHCA or 2,5-dihydroxy-benzoic acid (DHB) were used as matrices. It has been reported that a mixture comprising of 2,6-dihydroxy-acetophenone (DHAP) and di-ammonium hydrogen citrate (DAHC) is a suitable matrix for the MALDI analysis of a series of chemically labile compounds (Gorman et al, 1996). Using this "cooler" matrix, we eventually succeeded in obtaining molecular ions of the phosphorylated histidine-containing peptides in reflectron mode, although abundant ions were still detected due to dephosphorylation (Fig.…”

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

“…Comparison of DHAP, DHB, and CHCA Matrices-We used DHAP as the matrix because it is a "cooler" matrix than CHCA (30) and resulted in less PSD at the relatively high fixed laser energy (350 J/pulse) of our MALDI Q-TOF instrument. Fig.…”

The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

“…Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed with a Bruker Reflex mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) operated exclusively in the reflectron mode as described previously (25).…”

“…Peptides were diluted in 30% (v/v) aqueous acetonitrile containing 0.1% (v/v) trifluoracetic acid and 2 l of a matrix comprised of 2,6-dihydroxyacetophenone containing diammonium hydrogen citrate (25) prior to deposition of 0.5-1 l onto a stainless steel target. Reduction was achieved by adding diammonium hydrogen citrate to the diluted peptide solutions to a final concentration of 0.2 M followed by TCEP to a final concentration of 10 mM and incubation of the mixture for 20 min at 65°C.…”

“…Reduction was achieved by adding diammonium hydrogen citrate to the diluted peptide solutions to a final concentration of 0.2 M followed by TCEP to a final concentration of 10 mM and incubation of the mixture for 20 min at 65°C. A 2-l aliquot of the reduction mixture was mixed with 2 l of a 10 mg/ml solution of 2,6-dihydroxyacetophenone in 50% (v/v) ethanol/ acetonitrile and applied to the sample target of the mass spectrometer (25).…”