Manipulation of temperature to improve solubility of hydrophobic proteins and cocrystallization with matrix for analysis by MALDI-TOF mass spectrometry.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) requires cocrystallization of analyte with a large excess of matrix, which must be mutually soluble in a solvent that encourages crystal growth upon evaporation. MALDI-MS of hydrophobic proteins can be difficult, because they tend to aggregate in polar solutions. High concentrations of denaturants and salts are often employed to combat protein aggregation, but this can result in signal suppression. By using various organic cosolvent systems and matrixes at different protein:matrix ratios, we were able to use MALDI-TOFMS to detect four bacterially expressed hydrophobic proteins comprising alanine-rich mutants of the basic region/leucine zipper protein (bZIP) GCN4. By manipulating sample temperature, we were able to maintain protein solubility. Protein aggregation was suppressed when mixing the protein and matrix solutions at 4 degrees C prior to warming to 37 degrees C, following the temperature-leap technique described by Xie and Wetlaufer (Protein Sci. 1996, 5, 517-523), who used this method to renature bovine carbonic anhydrase II. Manipulation of temperature encouraged our hydrophobic proteins to adopt conformations leading to the nonaggregating state, and solubility was maintained even when the concentration of denaturant was reduced from 4 M to 400 mM. The temperature-leap tactic was critical for maintaining protein solubility, preventing signal suppression normally seen with higher concentrations of salts, allowing for generation of superior spectra, and should prove applicable to other systems prone to aggregation.
Bird
Gregory H
GH
Department of Chemistry, University of Pittsburgh, Pennsylvania 15260, USA.
Lajmi
Ajay R
AR
Shin
Jumi A
JA
D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors; D003460:Crystallization; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry; D051790:G-Box Binding Factors; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D051381:Rats; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000592:standards; D013696:Temperature; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D011506:Proteins; D014157:Transcription Factors