Direct characterization of overproduced proteins by native mass spectrometry

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Abstract

Proteins derived by recombinant technologies must be characterized to ensure quality, consistency and optimum production. These properties are usually assayed following purification procedures that are time-consuming and labor-intensive. Here we describe a native mass spectrometry approach, known as direct-MS, for rapid characterization of intact overexpressed proteins immediately from crude samples. In describing this protocol, we discuss the multiple applications of the method, and outline the necessary steps required for sample preparation, data collection and interpretation of results. We begin with the sample preparation workflows, which are relevant for either recombinant proteins produced within bacteria, those analyzed straight from crude cell lysate, or secreted proteins generated in eukaryotic expression systems that are assessed directly from the growth culture medium. We continue with the mass acquisition steps that enable immediate definition of properties such as expressibility, solubility, assembly state, folding, overall structure, stability, post-translational modifications, and associations with biomolecules. We demonstrate the applicability of the method through the characterization of a computationally designed toxin-anti toxin heterodimer, activity and protein interaction determination of a regulatory protein and detailed glycosylation analysis of a designed intact antibody. Overall, we describe a simple and rapid protocol that is relevant to both prokaryotic and eukaryotic expression systems that can be carried out on multiple mass spectrometers such as Orbitrap and QTOF-based platforms that enable intact protein detection. The entire procedure takes between 30 minutes to several hours, from sample collection to data acquisition, depending on the depth of MS analysis.

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Ion mobility-mass spectrometry analysis of large protein complexes.

Brandon T Ruotolo, Justin L. P. Benesch, Alan M. Sandercock … (2008)

Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility-mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility-mass spectrometry.

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Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry.

Helena Hernández, Carol V. Robinson (2007)

The growing number of applications to determine the stoichiometry, interactions and even subunit architecture of protein complexes from mass spectra suggests that some general guidelines can now be proposed. In this protocol, we describe the necessary steps required to maintain interactions between subunits in the gas phase. We begin with the preparation of suitable solutions for electrospray (ES) and then consider the transmission of complexes through the various stages of the mass spectrometer until their detection. Subsequent steps are also described, including the dissociation of these complexes into multiple subcomplexes for generation of interaction networks. Throughout we highlight the critical experimental factors that determine success. Overall, we develop a generic protocol that can be carried out using commercially available ES mass spectrometers without extensive modification.

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Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: a critical review.

Emanual Maverakis, Kyoungmi Kim, Michiko Shimoda … (2015)

Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode "self" identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses.