Denaturation of α‐lactalbumin and ubiquitin studied by electrospray and laser spray

Abstract

Electrospray and laser spray mass spectra of human alpha-lactalbumin and bovine ubiquitin were studied, with an emphasis on the denaturation induced by laser spray. There were no remarkable differences in the electrospray and laser spray mass spectra for acidic and basic aqueous solutions of alpha-lactalbumin in positive and negative modes of operations. This originates from the fact that this protein is tightly folded with four disulfide bonds. For ubiquitin, however, denaturation was induced by laser spray for the positive mode of operation and the [M+nH](n+) with a maximum of n = 13 was observed, i.e., all the acidic amino acid residues are fully neutralized (protonated). In contrast, the laser-induced denaturation was not observed for the negative mode of operation, i.e., denaturation of ubiquitin is largely suppressed in the negatively charged liquid droplets. The marked difference observed in the positive and negative modes of operations for ubiquitin is ascribed to the difference in the susceptibility of side-chain/main-chain interactions in the positive-ion excess and in the negative-ion excess liquid droplets. That is, the interactions between the basic residues and main-chain amide carbonyl groups (-NH(3) (+)***O=C< or -NH(2)***O=C<) which play an important role in stabilizing the protein structures are not so affected in the negative mode of operation but are weakened in the positive mode of operation.

Protein isolation through impact desolvation of electrosprayed microdroplets (IDEM): Molecular dynamics simulation

“…On the other hand, denaturation was found at positive mode operation for ubiquitin. In contrast, for both ␣-lactalbumin and ubiquitin laser-induced denaturation was not observed for the negative mode operations of laser spray as well as electro spray [21].…”

“…The role of jets and filaments in the electrospray process has been extensively investigated on the macroscopic scale [18][19][20]. For example, recent results of acidic and basic aqueous solutions of ␣-lactalbumin in positive and negative mode operations has found no remarkable differences in the laser and electro spray process [21]. On the other hand, denaturation was found at positive mode operation for ubiquitin.…”

Thermal unfolding of proteins probed by laser spray mass spectrometry

“…This is consistent with our previous report,25, 28, 31 in which the ion abundances generated by laser spray were found to improve by several orders of magnitude compared with those obtained by conventional ESI. In laser spray, thermal heating is completed instantaneously during the laser irradiation and no fragmentation is observed for the analyzed biomolecules, even when a laser beam with higher laser power (∼2.0 W) irradiates at the tip of the stainless‐steel capillary 30–34. These are the advantages of laser spray in stability analysis of biomolecules in solution.…”

“…Details of the instrumentation have already been published 25–34. In brief, a sample solution was introduced into a stainless‐steel capillary (i.d.…”

“…Laser spray, which was developed by Hiraoka and coworkers,25–34 is a hybrid of three basic techniques for the generation of gaseous ions from the condensed phase, i.e., energy‐sudden activation, nebulization and the action of an electric field. Laser spray mass spectrometry can faithfully reflect the solution‐phase characteristics of biomolecules.…”