title: Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.
abstract: Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.
authors.affiliation: Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang, Jiangxi 330013, P.R. China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang, Jiangxi 330013, P.R. China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Lu
authors.affiliation: Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang, Jiangxi 330013, P.R. China.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Chingin
authors.affiliation: Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang, Jiangxi 330013, P.R. China.
authors.forename: Huanwen
authors.initials: H
authors.lastname: Chen
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002645:Chickens; D006736:Horses; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry*; D055550:Protein Stability; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000596:Amino Acids; D009211:Myoglobin; D009113:Muramidase