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

✍ Scribed by Jeffrey J. Gorman; Bettina L. Ferguson; Thuong B. Nguyen

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 841 KB
• Volume: 10
• Category: Article
• ISSN: 0951-4198
• DOI: 10.1002/(sici)1097-0231(19960331)10:5<529::aid-rcm522>3.0.co;2-9

✦ Synopsis

Several peptides were shown to undergo fragmentation during matrix-assisted laser desorptionlionization timeof-flight mass spectrometry to a degree which complicated their analysis using a-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 &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 Pelimination of phosphate. It was demonstrated that the fragility exhibited by these peptides in CHCA, including &elimination of phosphate from serine, was not evident with a matrix comprising 2,6-dihydroxyacetophenone @HAP) and di-ammonium hydrogen citrate @AHC). The DHAPIDAHC matrix was also adapted for direct analysis of peptides from an acidic reducing milieu containing tris(2-carboxyethy1)phosphine. The molecular weight of equine cytochrome c was determined with a relatively high degree of accuracy (experimental Mr= 12360.2i1.4 Da compared to the theoretical Mr=12360.W Da) using DHAP/DAHC as a matrix for reflector mode analysis. ' 9

EXPERIMENTAL

Peptides and proteins Angiotensin 11, adrenocorticotropic hormone residues 18-39 (ACTH 18-39), bovine insulin, equine cytochrome c and equine myoglobin were purchased from the Sigma-Aldrich (Castle Hill, NSW, Australia). These peptides and proteins were dissolved in 0.1 % (v/v) aqueous trifluoroacetic acid at 100 pmol/pL and stored at -20 "C prior to CCC 09514198/96/050529-08