Abstract
Biological mass spectrometry (MS) encompasses a range of methods for characterizing proteins and other biomolecules. MS is uniquely powerful for the structural analysis of endogenous protein complexes, which are often heterogeneous, poorly abundant, and refractive to characterization by other methods. Here, we focus on how biological MS can contribute to the study of endogenous protein complexes, which we define as complexes expressed in the physiological host and purified intact, as opposed to reconstituted complexes assembled from heterologously expressed components. Biological MS can yield information on complex stoichiometry, heterogeneity, topology, stability, activity, modes of regulation, and even structural dynamics. We begin with a review of methods for isolating endogenous complexes. We then describe the various biological MS approaches, focusing on the type of information that each method yields. We end with future directions and challenges for these MS-based methods.
Highlights
Biological mass spectrometry (MS) encompasses a range of methods for characterizing proteins and other biomolecules
Rather than focusing on technical aspects of specific MS techniques, which are detailed in many excellent reviews cited here[4−6] and below, we focus on the compositional and structural insights that can be gained from biological MS and compare how different MS methods can yield this information
Different types of chromatographic separations are an important component of isolating endogenous complexes; here we focus on separation methods that typically constitute part of the MS analytical workflow, acknowledging that they overlap to some extent
Summary
The information extracted from MS experiments is limited by the scale and quality of the input samples. Many MS modalities can be applied to crude biological samples, as discussed below, typically assemblies must be extracted from cells or tissues and purified for MS analysis. For bottom-up proteomics analysis (see section 3.3.1), because proteins are digested and separated on a column, the complex does not need to be in an MS compatible buffer. For chemical footprinting methods (see section 3.3.2), complexes should be in a native state for the foot-printing step but are denatured and digested for MS analysis, relaxing complex purification requirements. For cross-linking MS with disuccinimidyl dibutyric urea, buffers containing primary amines should be avoided because they will react with the cross-linkers.[13] For native and top-down MS (see section 3.4), complexes should be eluted in an assembled, native state and exchanged into MS compatible buffers, as detailed in ref 14. Salts that can maintain physiological ionic strength and pH while remaining compatible with MS, including ammonium acetate, ethylene diammonium diacetate buffer, and others
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