Estimating Interprotein Pairwise Interaction Energies in Cell Lysates from a Single Native Mass Spectrum

Abstract

A powerful method to determine the energetic coupling between amino acids is double mutant cycle analysis. In this method, two residues are mutated separately and in combination and the energetic effects of the mutations are determined. A deviation of the effect of the double mutation from the sum of effects of the single mutations indicates that the two residues are interacting directly or indirectly. Here, we show that double mutant cycle analysis by native mass spectrometry can be carried out for interactions in crude Escherichia coli cell extracts, thereby obviating the need for protein purification and generating binding isotherms. Our results indicate that intermolecular hydrogen bond strengths are not affected by the more crowded conditions in cell lysates.

High intracellular stability of the spidroin N‐terminal domain in spite of abundant amyloidogenic segments revealed by in‐cell hydrogen/deuterium exchange mass spectrometry

“…However, this approach has limitations compared to nano-electrospray ionization (nESI) which allows top-down sequencing of the labelled proteins. Capitalizing on recent developments in the mass spectrometric analysis of overexpressed proteins directly from bacterial lysates [31,32], we developed a strategy where bacteria containing the overexpressed protein are diluted in > 100-fold excess of deuterated buffer to initiate labelling, while maintaining the physiological pH range to avoid distortion of the pH homeostasis of the bacteria. Deuterium penetration of E. coli under standard conditions occurs on the timescale of seconds, as judged by NMR and MS measurements [30,33].…”

Insight into the Autosomal-Dominant Inheritance Pattern of SOD1-Associated ALS from Native Mass Spectrometry

“…It also obviates the need to determine the values of the individual equilibrium constants, which is often laborintensive and error-prone. Importantly, coupling constants determined for crude cell lysates and purified proteins were found to be similar (13), thereby indicating that our approach does not perturb the equilibrium distribution in vivo of the various co-existing species.…”

“…This is possible because native MS is based on the ability to transfer protein complexes to the gas phase without disrupting them (10)(11)(12). Moreover, such transfer can take place from crude cell lysates (13). This approach (Fig.…”

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.…”