Effect of trifluoroacetic acid on the reduction of disulfide bridges in peptides analyzed by fast‐atom bombardment mass spectrometry

Abstract

Some of the factors that influence the reduction of disulfide-containing peptides under fast-atom bombardment have been investigated using two neurohormonal peptides that include disulfide bridges in their structures. Deaminoarginine-vasopressin (DAVP) and arginine-vasopressin (AVP) have been analyzed as their acetate and trifluoroacetate salts. Results obtained in a thioglycerol matrix indicate that the peptides analyzed as their acetate salts are completely reduced under bombardment, whereas the trifluoroacetate salts show little evidence of reduction. Addition of trifluoroacetic acid to the acetate sample prior to bombardment inhibits reduction whereas addition after bombardment shows no effect on the reduction, thereby indicating the irreversibility of the process. Time-monitoring experiments conducted with the acetate salts of DAVP and AVP in common matrices such as thioglycerol, dithiothreitol + diethioerythritol, glycerol, hydroxyethyldisulfide and nitrobenzyl-alcohol demonstrate an important effect of the chemical nature of the matrix on reduction. In matrices containing thiol groups, the reduction is extensive, whereas it is almost suppressed in matrices such as hydroxyethyldisulfide and nitrobenzylalcohol. However, the addition of trifluoroacetic acid to all of these matrices essentially eliminates reduction and provides measured isotopic peak ratios that are in agreement with theoretically calculated values for these peptides.

Evaluation of the true effect of experimental parameters on the reduction / oxidation processes observed in fast-atom bombardment/liquid secondary spectrometry

“…and Back , will take two average values, thus minimizing the errors on background assessment and on the values of redox processes. The ion intensities measured in the molecular ion region can be simulated according to eqs [11][12][13][14][15][16][17][18], which include all of the variables of interest. It is the I k values, which include the contributions from background, natural isotopic abundances, and the oxidized/reduced species accordingly, that the program tries to best match with observed peak inten-sities, The model uses eqs 12-17 to estimate values for the processes and eqs 11-18 to optimize them.…”

“…Instrumental parameters associated with the FAB/LSIMS source conditions may also play an im-portant role in determining the extent of reduction observed. Those parameters that have been studied include time of irradiation [8,14,16,17,23]; primary beam energy [15,20,28); and bombarding beam density (flux) [12,15,20,23,28]. Wirth et al [8] observed a time dependence in their FAB analysis (Xe O primary beam) of azo-group-eontaining peptides.…”

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration

“…We, however, have not observed a difference in nanoparticle structure when PEG-S-POD was equilibrated in either buffer (data not shown). However, TFA has been found to inhibit reduction of disulfide bonds 22 . To determine whether the use of either TFA or AmAc as a counterion for equilibration of PEG-SS-POD could inhibit reduction of the disulfide bond, we incubated PEG-SS-POD equilibrated in each buffer with β-mercaptoethanol (Figure 1D).…”

The role of a liquid matrix in controlling FAB-induced fragmentation

“…Addition of trifluoroacetic acid (TFA) to glycerol-thioglycerol matrix mixture also assists in maintaining the integrity of the disulfide bond. 27 In 1987, we reported in detail the FAB-induced fragmentation behavior of several C-terminal-extended enkephalins using glycerol as the matrix." The foremost process of sequence ion formation in the positive-ion FAB was fission of the amino-alkyl bond of the peptide backbone to yield z-type C-terminal sequence ions.…”