Use of 2,6-dihydroxyacetophenone for analysis of fragile peptides, disulphide bonding and small proteins by matrix-assisted laser desorption/ionization.
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.
Gorman
J J
JJ
Biomolecular Research Institute, Parkville, Victoria, Australia.
Ferguson
B L
BL
Nguyen
T B
TB
D000098:Acetophenones / Q000737:chemistry*; D000595:Amino Acid Sequence; D002951:Citrates / Q000737:chemistry; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000098:Acetophenones; D002951:Citrates; D004220:Disulfides; D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins; D000644:Quaternary Ammonium Compounds; C019339:2,6-dihydroxyacetophenone