Abstract
Biomolecular mass spectrometry has matured strongly over the past decades and has now reached a stage where it can provide deep insights into the structure and composition of large cellular assemblies. Here, we describe a three-tiered hybrid mass spectrometry approach that enables the dissection of macromolecular complexes in order to complement structural studies. To demonstrate the capabilities of the approach, we investigate ribosomes, large ribonucleoprotein particles consisting of a multitude of protein and RNA subunits. We identify sites of sequence processing, protein post-translational modifications, and the assembly and stoichiometry of individual ribosomal proteins in four distinct ribosomal particles of bacterial, plant and human origin. Amongst others, we report extensive cysteine methylation in the zinc finger domain of the human S27 protein, the heptameric stoichiometry of the chloroplastic stalk complex, the heterogeneous composition of human 40S ribosomal subunits and their association to the CrPV, and HCV internal ribosome entry site RNAs.
Highlights
Biomolecular mass spectrometry has matured strongly over the past decades and has reached a stage where it can provide deep insights into the structure and composition of large cellular assemblies
Before describing the in-depth analysis of the ribosomal particles by our three-tiered mass spectrometry (MS) approach, we first describe some novel workflows, hardware and software we used for top-down LC-MS/MS making use of a recently introduced mass analyzer, the OrbitrapTMHF-X27
When full MS spectra are acquired to determine which proteoforms are selected for subsequent top-down sequencing events, there is an information redundancy that comes from the repeated selection and fragmentation of the different charge states of the same proteoform
Summary
Biomolecular mass spectrometry has matured strongly over the past decades and has reached a stage where it can provide deep insights into the structure and composition of large cellular assemblies. Even with structures of ribosomes from many kingdoms of life and different organelles resolved[12,13,14,15], small but potentially important features of ribosomal particles have been mostly overlooked These features, including specific posttranslational modifications (PTMs), sequence variations, binding of protein cofactors or sub-stoichiometric presence of ribosomal proteins, can be elusive to standard structural biology techniques and require the use of complementary approaches, such as mass spectrometry (MS). Bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS), a MS technique commonly used in proteomics research, provides the ability to identify and quantify the ribosomal proteins and their PTMs16 It can determine the presence of ribosomeinteracting factors, which have remained bound to the ribosomal particles during their purification[17]. We demonstrate how this three-tiered MS approach can provide in-depth characterization of four distinct ribosomal particles: E. coli cytosolic 70S ribosomes (Ec70S), chloroplastic 70S ribosomes from spinach (So70S), and human cytosolic 40S (Hs40S) and 60S (Hs60S) ribosomal subunits (Table 1)
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