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Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes.

ABSTRACT: With an overarching goal of characterizing the structure of every protein within a cell, identifying its interacting partners, and quantifying the dynamics of the states in which it exists, key developments are still necessary to achieve comprehensive native proteomics by mass spectrometry (MS). In practice, much work remains to optimize reliable online separation methods that are compatible with native MS and improve tandem MS (MS/MS) approaches with respect to when and how energy is deposited into proteins of interest. Herein, we utilize native capillary zone electrophoresis coupled with MS to characterize the proteoforms in the Escherichia coli 70S ribosome. The capabilities of 193 nm ultraviolet photodissociation (UVPD) to yield informative backbone sequence ions are compared to those of higher-energy collisional dissociation (HCD). To further improve sequence coverage values, a multistage MS/MS approach is implemented involving front-end collisional activation to disassemble protein complexes into constituent subunits that are subsequently individually isolated and activated by HCD or UVPD. In total, 48 of the 55 known E. coli ribosomal proteins are identified as 84 unique proteoforms, including 22 protein-metal complexes and 10 protein-protein complexes. Additionally, mapping metal-bound holo fragment ions resulting from UVPD of protein-metal complexes offers insight into the metal-binding sites.

SUBMITTER: Mehaffey MR

PROVIDER: S-EPMC7788560 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×10(5) in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10(-18)-10(-20)mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

Dataset Information

Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes.

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