Native Mass Spectrometry of Recombinant Proteins from Crude Cell Lysates

Abstract

Determining the properties of proteins prior to purification saves time and labor. Here, we demonstrate a native mass spectrometry approach for rapid characterization of overexpressed proteins directly in crude cell lysates. The method provides immediate information on the identity, solubility, oligomeric state, overall structure and stability, as well as ligand binding, without the need for purification.

Surface-Induced Dissociation: An Effective Method for Characterization of Protein Quaternary Structure

“…Direct introduction of cell lysates using static nano-spray for detection of overexpressed protein complexes has been reported, which simplifies the protein purification steps. 232,233 The long-term goal for such studies would be to study single cells. An alternative approach to this is online desalting.…”

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

“…Excellent work has recently demonstrated that intracellular and secreted proteins can be analyzed by native MS after overexpression via a so-called "direct MS" method if nonvolatile molecules are excluded in the resuspension solution and are first removed by washing the cell pellets. [50][51][52][53] The direct MS method is tailored for the analysis of cell lysates and supernatants, making it suitable for monitoring protein overexpression. In case additional purification steps are required due to low expression or weak ionization, this method typically cannot be used without a buffer exchange step due to the necessity of introducing non-volatiles (i.e.…”

“…Recent work has demonstrated the use of native MS to directly analyze cell lysates or supernatants to monitor protein expression and biomolecular interactions. [50][51][52] Generally, these methods require washing or buffer exchange steps prior to analysis of the sample by nano ESI. We therefore envision that these "direct MS" methods are complementary to the OBE nMS method as OBE will allow for automated buffer exchange of the cell lysate, bypassing the offline washing and/or buffer exchange steps.…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…MS-compatible detection methods enable MS analysis after electrophoresis (Raynal et al 2014). Despite such analysis are usually performed after purification, Gan et al (2017) reported a native MS approach that allows the characterization of overexpressed recombinant proteins directly in crude E. coli lysates, allowing obtaining information on its identity, solubility, oligomeric state, overall structure, and stability without purification. Cells were lysed in a buffer supplemented with 1 M ammonium acetate to ensure compatibility with MS. Spectra were acquired for distinct proteins with molecular weights ranging from 19 to 47 kDa, and revealed highly resolved peaks, narrow charge state distributions, and the anticipated stoichiometry, thereby confirming that at least for these proteins, purification is not a prerequisite (Gan et al 2017).…”