Mowei Zhou

Protein Subunits Released by Surface Collisions of Noncovalent Complexes: Nativelike Compact Structures Revealed by Ion Mobility Mass Spectrometry†

The quaternary structure of proteins determines their biological function, and a majority of proteins exist as oligomers in vivo with miscellaneous architectures. Mass spectrometry (MS) can be applied to study the stoichiometry and interactions of protein complexes by molecular weight measurements under gentle instrumental conditions where noncovalent interactions are preserved in the gas phase. Recently, there have been extensive efforts to also utilize ion mobility (IM) techniques combined with MS for structural studies of noncovalent biological complexes, including virus assembly pathways, because IM provides conformational information, which is not accessible by MS, for these gas-phase ions. Experimentally measured collisional cross sections (CCSs) from IM can serve as constraints for architecture determination by molecular modeling. Additionally, tandem MS can be used to dissociate gas-phase complexes. A protein assembly would ideally dissociate into various noncovalent subcomplexes, and the topology of the original complex could be derived by piecing together all the subcomplex products. The common tandem MS method, collision induced dissociation (CID), involves activation of the complexes by collision with neutral gas atoms or molecules. Typically, CID results in an “asymmetric” dissociation into highly charged monomers and complementary (n 1)-mers (although a few exceptions have been reported), and studies have suggested that unfolding of protein complexes occurs in CID. It is therefore difficult to relate the CCS measurements of CID product ions to the complexes native structure. Tandem MS can alternatively be achieved by surface induced dissociation (SID) where the complexes collide with a surface target. Previous research in our group has shown that several protein complexes dissociate in a more “symmetric” manner with SID than by CID, and have charge distributed more proportionally to the mass. We hypothesized that dissociation might occur in the absence of gradual monomer unfolding for SID because activation by SID is a single-step, higher-energy deposition, fast process that is different from the multistep, slower CID process. SID has recently been applied to determining the quaternary structure of a heterohexameric protein with information from subunit product ions such as heterotrimers unique to SID. We present herein the first IM measurements on the SID products of several protein complexes, along with comparison to CID products, by using a modified quadrupole/IM/time-offlight (Q/IM/TOF) instrument. Briefly, the precursor ions are dissociated by CID or SID cells placed in front of the IM cell. The product ions are subsequently separated based on their size, shape, and charge under the influence of a continuous series of electrical pulses and friction with neutral gas in the IM cell. The drift times of the ions are recorded, with larger and lower-charged ions experiencing longer drift times. Experimental CCSs can be derived from the measured drift times and mass-to-charge-ratios. Theoretical CCSs can be calculated from crystal structures. Nativelike ions should have experimental CCSs similar to the crystal structure, whereas unfolded ions are expected to show larger CCS values because of an increased surface area. We first examined the remaining undissociated pentamer precursor of C-reactive protein (CRP) after activation by either CID or SID. Triethylammonium acetate (TEAA) was added in the electrospraying buffer, which has been reported as a charge reducing additive to increase the stability of protein complexes in the gas phase. Without TEAA no remaining precursor could be observed in SID, even at low acceleration voltages. The addition of TEAA did not cause any remarkable structural change of the protein as the CCSs of the precursor did not change after charge reduction. The + 18 precursor of CRP was selected and activated. Examination of precursor CCSs at various SID acceleration voltages (20–50 V) reveals that most of the CRP pentamer dissociated without extensive increase in CCS of the remaining precursor. In CID, however, the CCS of the undissociated CRP pentamer first decreased at low acceleration voltages and then increased considerably above its dissociation threshold of about 80 V. It reached a stable unfolding intermediate with CID around 100 V, where the CCS does not additionally increase with increasing CID acceleration voltages (data not shown). It is impractical to determine one acceleration voltage at which the amounts of the internal energies deposited in CID and SID are identical because of different mechanisms and complications from the physical properties of large protein complexes. Nonetheless, we show here a representative comparison between CID at 100 V (Figure 1, top right) and SID at 40 V (Figure 1, bottom [*] M. Zhou, Dr. S. Dagan, Prof. V. H. Wysocki Department of Chemistry and Biochemistry, University of Arizona 1306 E. University Blvd., Tucson, AZ (USA) E-mail: vwysocki@email.arizona.edu [] Permanent address: Israel Institute for Biological Research (IIBR) POB 19, Ness Ziona 74100 (Israel)

Dissecting the Large Noncovalent Protein Complex GroEL with Surface-Induced Dissociation and Ion Mobility–Mass Spectrometry

Tandem mass spectrometry is a tool to dissect noncovalent protein complexes into smaller substructures for quaternary structure analysis. The commonly used activation method, collision induced dissociation (CID), often provides limited structural information from the typical dissociation pattern where unfolded monomers are ejected from the protein complex. In contrast, surface-induced dissociation (SID) has been shown to be very effective at dissociating protein complexes with less unfolding than CID. We present here SID of a large noncovalent tetradecamer protein, GroEL (801 kDa). A wide variety of products, including heptamers representative of the native topology, are released from the precursor upon SID, significantly different from the ubiquitous monomer ejection in CID. Enhanced dissociation into heptamers is observed when the charge states of the GroEL precursor are reduced by adding triethylammonium acetate into the spraying buffer. Ion mobility is utilized after SID to separate products overlapping in m/z to simplify the SID spectra. Compact heptamers from the charge-reduced tetradecamer are clearly distinguished from other overlapping species. SID can be very useful for quaternary structure studies of large noncovalent protein complexes, as manifested by the GroEL data where the tetradecamer dissociates into heptamers, reflecting the native topology of the complex.

Surface Induced Dissociation: Dissecting Noncovalent Protein Complexes in the Gas phase

The quaternary structures of proteins are both important and of interest to chemists, because many proteins exist as complexes in vivo, and probing these structures allows us to better understand their biological functions. Conventional structural biology methods such as X-ray crystallography and nuclear magnetic resonance provide high-resolution information on the structures of protein complexes and are the gold standards in the field. However, other emerging biophysical methods that only provide low-resolution data (e.g. stoichiometry and subunit connectivity) on the structures of the protein complexes are also becoming more important to scientists. Mass spectrometry is one of these approaches that provide lower than atomic structural resolution, but the approach is higher throughput and provides not only better mass information than other techniques but also stoichiometry and topology. Fragile noncovalent interactions within the protein complexes can be preserved in the gas phase of MS under gentle ionization and transfer conditions. Scientists can measure the masses of the complexes with high confidence to reveal the stoichiometry and composition of the proteins. What makes mass spectrometry an even more powerful method is that researchers can further isolate the protein complexes and activate them in the gas phase to release subunits for more structural information. The caveat is that, upon gas-phase activation, the released subunits need to faithfully reflect the native topology so that useful information on the proteins can be extracted from mass spectrometry experiments. Unfortunately, many proteins tend to favor unfolding upon collision with neutral gas (the most common activation method in mass spectrometers). Therefore, this typically results in limited insights on the quaternary structure of the precursor without further manipulation of other experimental factors. Scientists have observed, however, that valuable structural information can be obtained when the gas-phase proteins are activated by collision with a surface. Subcomplexes released after surface collision are consistent with the native quaternary structure of several protein systems studied, even for a large chaperone protein, GroEL, that approaches megadalton mass. The unique and meaningful data generated from surface induced dissociation (SID) have been attributed to the fast and energetic activation process upon collision with a massive target, the surface. In this Account, we summarize our SID studies of protein complexes, with emphasis on the more recent work on the combination of ion mobility (IM) with SID. IM has gained popularity over the years not only as a gas-phase separation technique but also as a technique with the ability to measure the size and shape of the proteins in the gas phase. Incorporation of IM before SID allows different conformations of a protein to be separated and examined individually by SID for structural details. When IM is after SID, the cross sections of the SID products can be measured, providing insight on the dissociation pathways, which may mimic disassembly pathways. Furthermore, the separation by IM greatly reduces the peak overlapping (same m/z) and coalescence (merging) of SID products, improving the resolving power of the method. While there are still many unanswered questions on the fundamental properties of gas-phase proteins and a need for further research, our work has shown that SID can be a complementary gas-phase tool providing useful information for studying quaternary structures of noncovalent protein complexes.

Surface-Induced Dissociation of Ion Mobility-Separated Noncovalent Complexes in a Quadrupole/Time-of-Flight Mass Spectrometer

A custom in-line surface-induced dissociation (SID) device has been incorporated into a commercial ion mobility quadrupole/time-of-flight mass spectrometer in order to provide an alternative and potentially more informative activation method than the commonly used collision-induced dissociation (CID). Complicated sample mixtures can be fractionated by ion mobility (IM) and then dissociated by CID or SID for further structural analysis. Interpretation of SID spectra for cesium iodide clusters was greatly simplified with IM prior to dissociation because products originating from different precursors and overlapping in m/z but separated in drift time can be examined individually. Multiple conformations of two protein complexes, source-activated transthyretin tetramer and nativelike serum amyloid P decamer, were separated in ion mobility and subjected to CID and SID. CID spectra of the mobility separated conformations are similar. However, drastic differences can be observed for SID spectra of different conformations, implying different structures in the gas phase. This work highlights the potential of utilizing IM-SID to study quaternary structures of protein complexes and provides information that is complementary to our recently reported SID-IM approach.

Surface-Induced Dissociation Mass Spectra as a Tool for Distinguishing Different Structural Forms of Gas-Phase Multimeric Protein Complexes.

One attractive feature of ion mobility mass spectrometry (IM-MS) lies in its ability to provide experimental collision cross section (CCS) measurements, which can be used to distinguish different conformations that a protein complex may adopt during its gas-phase unfolding. However, CCS values alone give no detailed information on subunit structure within the complex. Consequently, structural characterization typically requires molecular modeling, which can have uncertainties without experimental support. One method of obtaining direct experimental evidence on the structures of these intermediates is utilizing gas-phase activation techniques that can effectively dissociate the complexes into substructures while preserving the native topological information. The most commonly used activation method, collision-induced dissociation (CID) with low-mass target gases, typically leads to unfolding of monomers of a protein complex. Here, we describe a method that couples IM-MS and surface-induced dissociation (SID) to dissociate the source-activated precursors of three model protein complexes: C-reactive protein (CRP), transthyretin (TTR), and concanavalin A (Con A). The results of this study confirm that CID involves the unfolding of the protein complex via several intermediates. More importantly, our experiments also indicate that retention of similar CCS between different intermediates does not guarantee retention of structure. Although CID spectra (at a given collision energy) of source-activated, mass-selected precursors do not distinguish between native-like, collapsed, and expanded forms of a protein complex, dissociation patterns and/or average charge states of monomer products in SID of each of these forms are unique.

Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

AbstractIon mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer. Figureᅟ

Structural analysis of activated SgrAI-DNA oligomers using ion mobility mass spectrometry.

SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits self-modulation of DNA cleavage activity and sequence specificity. Previous studies have shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of the SgrAI-DNA complex as well as the basic building block of the oligomers have not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI-DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD) with one SgrAI dimer bound to two precleaved duplex DNA molecules each containing one-half of the SgrAI primary recognition sequence. The oligomers contain variable numbers of DBDs with as many as 19 DBDs. Observation of the large oligomers shows that nanoelectrospray ionization (nano-ESI) can preserve the proposed activated form of an enzyme. Finally, the collision cross section of the SgrAI-DNA oligomers measured by IM-MS was found to have a linear relationship with the number of DBDs in each oligomer, suggesting a regular, repeating structure.

Informed-Proteomics: open-source software package for top-down proteomics

Top-down proteomics, the analysis of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liquid chromatography–tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed. Here we present Informed-Proteomics, an open-source software suite for top-down proteomics analysis that consists of an LC-MS feature-finding algorithm, a database search algorithm, and an interactive results viewer. We compare our tool with several other popular tools using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up analysis.

Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry

As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biology and their roles in disease development have been the subject of intense study. Because multiple post-translational modifications (PTMs) along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here we used state-of-the-art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as a search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

Gene regulation by substoichiometric heterocomplex formation of undecameric TRAP and trimeric anti-TRAP

Significance Noncovalent interactions between proteins modulate their functions and occur widely in biological regulation. A large proportion of such regulatory proteins are homo-oligomeric, with multiple copies of a single polypeptide assembled into higher-order quaternary structures. Understanding the regulatory interactions between homo-oligomeric proteins is difficult because their periodic structural configuration may allow different modes of interaction with differing functions. We apply a powerful combination of analytical techniques to study the interaction between TRAP (trp RNA-binding attenuation protein), an 11-mer that regulates tryptophan metabolism by binding RNA, and its trimeric inhibitor protein anti-TRAP. We show that anti-TRAP condenses multiple TRAP oligomers into heterocomplexes, thereby blocking TRAP’s RNA-binding sites. These findings and our approach may have broad implications for other oligomeric regulatory proteins. The control of tryptophan production in Bacillus is a paradigmatic example of gene regulation involving the interplay of multiple protein and nucleic acid components. Central to this combinatorial mechanism are the homo-oligomeric proteins TRAP (trp RNA-binding attenuation protein) and anti-TRAP (AT). TRAP forms undecameric rings, and AT assembles into triskelion-shaped trimers. Upon activation by tryptophan, the outer circumference of the TRAP ring binds specifically to a series of tandem sequences present in the 5′ UTR of RNA transcripts encoding several tryptophan metabolism genes, leading to their silencing. AT, whose expression is up-regulated upon tryptophan depletion to concentrations not exceeding a ratio of one AT trimer per TRAP 11-mer, restores tryptophan production by binding activated TRAP and preventing RNA binding. How the smaller AT inhibitor prevents RNA binding at such low stoichiometries has remained a puzzle, in part because of the large RNA-binding surface on the tryptophan-activated TRAP ring and its high affinity for RNA. Using X-ray scattering, hydrodynamic, and mass spectrometric data, we show that the polydentate action of AT trimers can condense multiple intact TRAP rings into large heterocomplexes, effectively reducing the available contiguous RNA-binding surfaces. This finding reveals an unprecedented mechanism for substoichiometric inhibition of a gene-regulatory protein, which may be a widespread but underappreciated regulatory mechanism in pathways that involve homo-oligomeric or polyvalent components.