The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."

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.

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