RESUMO
Native ion mobility/mass spectrometry is well-poised to structurally screen proteomes but characterizes protein structures in the absence of a solvent. This raises long-standing unanswered questions about the biological significance of protein structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the unfolding of the protein ubiquitin in a solvent-free environment. Our data suggest that the folded, solvent-free ubiquitin observed by ion mobility/mass spectrometry exists in a largely native fold with an intact ß-grasp motif and α-helix. The ensemble of folded, solvent-free ubiquitin ions can be partitioned into kinetically stable subpopulations that appear to correspond to the structural heterogeneity of ubiquitin in solution. Time-resolved ion mobility/ion mobility/mass spectrometry measurements show that folded, solvent-free ubiquitin exhibits a strongly stretched-exponential time dependence, which simulations trace to a rugged energy landscape with kinetic traps. Unfolding rate constants are estimated to be approximately 800 to 20,000 times smaller than in the presence of water, effectively quenching the unfolding process on the time scale of typical ion mobility/mass spectrometry measurements. Our proposed unfolding pathway of solvent-free ubiquitin shares substantial characteristics with that established for the presence of solvent, including a polarized transition state with significant native content in the N-terminal ß-hairpin and α-helix. Our experimental and computational data suggest that (1) the energy landscape governing the motions of folded, solvent-free proteins is rugged in analogy to that of glassy systems; (2) large-scale protein motions may at least partially be determined by the amino acid sequence of a polypeptide chain; and (3) solvent facilitates, rather than controls, protein motions.
RESUMO
Top-down proteomics provides a straightforward approach to the level of proteoforms but remains technologically challenging. Using ion mobility spectrometry/mass spectrometry (IMS/MS) to separate top-down fragment ions improves signal/noise and dynamic range. Such applications, however, do not yet leverage the primary information obtained from IMS/MS, which is the characterization of the fragment ion structure by the measured momentum transfer cross sections. Here, we perform top-down analysis of intact proteins and assemblies using our tandem trapped ion mobility spectrometer/mass spectrometer (tTIMS/MS) and compile over 1400 cross section values of fragment ions. Our analysis reveals that most fragment ions exhibit multiple, stable conformations similar to those of intact polypeptides and proteins. The data further indicate that the conformational heterogeneity is strongly influenced by the amino acid sequences of the fragment ions. Moreover, time-resolved tTIMS/MS experiments reveal that conformations of top-down fragment ions can be metastable on the timescale of ion mobility measurements. Taken together, our analysis indicates that top-down fragment ions undergo a folding process in the gas phase and that this folding process can lead to kinetic trapping of intermediate states in ion mobility measurements. Hence, because the folding free energy surface of a polypeptide ion is encoded by its amino acid sequence and charge state, our analysis suggests that cross sections can be exploited as sequence-specific determinants of top-down fragment ions.
Assuntos
Espectrometria de Mobilidade Iônica , Proteínas , Espectrometria de Mobilidade Iônica/métodos , Íons/química , Peptídeos , Proteínas/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Ion mobility spectrometry/mass spectrometry (IMS/MS) is widely used to study various levels of protein structure. Here, we review the current state of affairs in tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS). Two different tTIMS/MS instruments are discussed in detail: the first tTIMS/MS instrument, constructed from coaxially aligning two TIMS devices; and an orthogonal tTIMS/MS configuration that comprises an ion trap for irradiation of ions with UV photons. We discuss the various workflows the two tTIMS/MS setups offer and how these can be used to study primary, tertiary, and quaternary structures of protein systems. We also discuss, from a more fundamental perspective, the processes that lead to denaturation of protein systems in tTIMS/MS and how to soften the measurement so that biologically meaningful structures can be characterised with tTIMS/MS. We emphasize the concepts underlying tTIMS/MS to underscore the opportunities tandem-ion mobility spectrometry methods offer for investigating heterogeneous samples.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas em Tandem , Espectrometria de Mobilidade Iônica/métodos , Íons/química , Proteínas , Espectrometria de Massas em Tandem/métodosRESUMO
RATIONALE: Tandem-ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem-ion mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes. METHODS: Here, we describe the coupling of the separation capabilities of tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis. RESULTS: We establish the feasibility of dissociating intact proteins by UV irradiation at 213 nm between the two TIMS devices in tTIMS/MS and at pressure conditions compatible with ion mobility spectrometry (2-3 mbar). We validate that the fragments produced by UVPD under these conditions result from a radical-based mechanism in accordance with prior literature on UVPD. The data suggest stabilization of fragment ions produced from UVPD by collisional cooling due to the elevated pressures used here ("UVnoD2"), which otherwise do not survive to detection. The data account for a sequence coverage for the protein ubiquitin comparable to recent reports, demonstrating the analytical utility of our instrument in mobility-separating fragment ions produced from UVPD. CONCLUSIONS: The data demonstrate that UVPD carried out at elevated pressures of 2-3 mbar yields extensive fragment ions rich in information about the protein and that their exhaustive analysis requires IMS separation post-UVPD. Therefore, because UVPD and tTIMS/MS each have been shown to be valuable techniques on their own merit in proteomics, our contribution here underscores the potential of combining tTIMS/MS with UVPD for structural proteomics.
RESUMO
Glycoproteins play a central role in many biological processes including disease mechanisms. Nevertheless, because glycoproteins are heterogeneous entities, it remains unclear how glycosylation modulates the protein structure and function. Here, we assess the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (tandem-TIMS/MS) to characterize the structure and sequence of the homotetrameric glycoprotein avidin. We show that (1) tandem-TIMS/MS retains native-like avidin tetramers with deeply buried solvent particles; (2) applying high activation voltages in the interface of tandem-TIMS results in collision-induced dissociation (CID) of avidin tetramers into compact monomers, dimers, and trimers with cross sections consistent with X-ray structures and reports from surface-induced dissociation (SID); (3) avidin oligomers are best described as heterogeneous ensembles with (essentially) random combinations of monomer glycoforms; (4) native top-down sequence analysis of the avidin tetramer is possible by CID in tandem-TIMS. Overall, our results demonstrate that tandem-TIMS/MS has the potential to correlate individual proteoforms to variations in protein structure.
Assuntos
Avidina/análise , Espectrometria de Mobilidade Iônica , Conformação Proteica , Espectrometria de Massas em TandemRESUMO
Ion mobility spectrometry-mass spectrometry (IMS-MS) determines momentum transfer cross sections of ions to elucidate their structures. Recent IMS methods employ electrodynamic fields or nonstationary buffer gases to separate ions. These methods require a calibration procedure to determine ion mobilities from the experimental data. This applies in particular to trapped IMS (TIMS), a novel IMS method with reported high resolving powers. Here, we report the first systematic assessment of the accuracy and the limitations of mobility calibration in TIMS. Our data show that the currently used TIMS calibration approach reproduces drift tube mobilities to approximately 1% (95th percentile). Furthermore, we develop a transferable and sample-independent calibration procedure for TIMS. The central aspects of our approach are (1) a calibration function derived from a solution to the Boltzmann transport equation and (2) calibration constants based on a Taylor expansion of instrument properties (TEIP). The key advantage of our calibration approach over current ones is its transferability: one equation and one set of parameters are sufficient to calibrate ion mobilities for various instrument settings, compound classes, or charge states. Our approach is transferable over time and sufficiently accurate (â¼1-2%) for structure-elucidation purposes. While we develop our calibration procedure specifically for TIMS, the approach we take is generic in nature and can be applied to other IMS systems.
RESUMO
There is currently a strong interest in the use of ion mobility spectrometry-mass spectrometry (IMS-MS) instrumentation for structural biology. In these applications, momentum transfer cross sections derived from IMS-MS measurements are used to reconstruct the three-dimensional analyte structure. Recent reports indicate that additional structural information can be extracted from measuring changes in cross sections in response to changes of the analyte structure. To further this approach, we constructed a tandem trapped IMS analyser (TIMS-TIMS) and incorporated it in a QqTOF mass spectrometer. TIMS-TIMS is constructed by coupling two TIMS analysers via an "interface region" composed of two apertures. We show that peptide oligomers (bradykinin) and native-like protein (ubiquitin) ions can be preserved through the course of an experiment in a TIMS-TIMS analyser. We demonstrate the ability to collisionally-activate as well as to trap mobility-selected ions, followed by subsequent mobility-analysis. In addition to inducing conformational changes, we show that we can fragment low charge states of ubiquitin at >1 mbar between the TIMS analysers with significant sequence coverage. Many fragment ions exhibit multiple features in their TIMS spectra, which means that they may not generally exist as the most stable isomer. The ability of TIMS-TIMS to dissociate mobility-selected protein ions and to measure the cross sections of their fragment ions opens new possibilities for IMS-based structure elucidation.
RESUMO
Key to native ion mobility/mass spectrometry is to prevent the structural denaturation of biological molecules in the gas phase. Here, we systematically assess structural changes induced in the protein ubiquitin during a trapped ion mobility spectrometry (TIMS) experiment. Our analysis shows that the extent of structural denaturation induced in ubiquitin ions is largely proportional to the amount of translational kinetic energy an ion gains from the applied electric field between two collisions with buffer gas particles. We then minimize the efficiency of the structural denaturation of ubiquitin ions in the gas phase during a TIMS experiment. The resulting "soft" TIMS spectra of ubiquitin are found largely identical to those observed on "soft" elevated-pressure ion mobility drift tubes and the corresponding calibrated cross sections are consistent with structures reported from NMR experiments for the native and A-state of ubiquitin. Thus, our analysis reveals that TIMS is useful for native ion mobility/mass spectrometry analysis.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Ubiquitina/química , ÍonsRESUMO
Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Piruvato Quinase/química , Piruvato Quinase/análise , Estreptavidina/química , Estreptavidina/análise , Toxina da Cólera/análise , Toxina da Cólera/química , Avidina/química , Avidina/análise , Proteínas/análise , Proteínas/químicaRESUMO
Ion activation methods carried out at gas pressures compatible with ion mobility separations are not yet widely established. This limits the analytical utility of emerging tandem-ion mobility spectrometers that conduct multiple ion mobility separations in series. The present work investigates the applicability of collision-induced dissociation (CID) at 1 to 3 mbar in a tandem-trapped ion mobility spectrometer (tandem-TIMS) to study the architecture of protein complexes. We show that CID of the homotetrameric protein complexes streptavidin (53 kDa), neutravidin (60 kDa), and concanavalin A (110 kDa) provides access to all subunits of the investigated protein complexes, including structurally informative dimers. We report on an "atypical" dissociation pathway, which for concanavalin A proceeds via symmetric partitioning of the precursor charges and produces dimers with the same charge states that were previously reported from surface induced dissociation. Our data suggest a correlation between the formation of subunits by CID in tandem-TIMS/MS, their binding strengths in the native tetramer structures, and the applied activation voltage. Ion mobility spectra of in situ-generated subunits reveal a marked structural heterogeneity inconsistent with annealing into their most stable gas phase structures. Structural transitions are observed for in situ-generated subunits that resemble the transitions reported from collision-induced unfolding of natively folded proteins. These observations indicate that some aspects of the native precursor structure is preserved in the subunits generated from disassembly of the precursor complex. We rationalize our observations by an approximately 100-fold shorter activation time scale in comparison to traditional CID in a collision cell. Finally, the approach discussed here to conduct CID at elevated pressures appears generally applicable also for other types of tandem-ion mobility spectrometers.
Assuntos
Proteínas , Concanavalina A/química , Proteínas/químicaRESUMO
Cellular processes are usually carried out collectively by the entirety of all proteins present in a biological cell, i.e. the proteome. Mass spectrometry-based methods have proven particularly successful in identifying and quantifying the constituent proteins of proteomes, including different molecular forms of a protein. Nevertheless, protein sequences alone do not reveal the function or dysfunction of the identified proteins. A straightforward way to assign function or dysfunction to proteins is characterization of their structures and dynamics. However, a method capable to characterize detailed structures of proteins and protein complexes in a large-scale, systematic manner within the context of cellular processes does not yet exist. Here, we discuss the potential of tandem-ion mobility / mass spectrometry (tandem-IM/MS) methods to provide such ability. We highlight the capability of these methods using two case studies on the protein systems ubiquitin and avidin using the tandem-TIMS/MS technology developed in our laboratory and discuss these results in the context of other developments in the broader field of tandem-IM/MS.
RESUMO
Characterizing structures of protein complexes and their disease-related aberrations is essential to understanding molecular mechanisms of many biological processes. Electrospray ionization coupled with hybrid ion mobility/mass spectrometry (ESI-IM/MS) methods offer sufficient sensitivity, sample throughput, and dynamic range to enable systematic structural characterization of proteomes. However, because ESI-IM/MS characterizes ionized protein systems in the gas phase, it generally remains unclear to what extent the protein ions characterized by IM/MS have retained their solution structures. Here, we discuss the first application of our computational structure relaxation approximation [Bleiholder, C.; et al. J. Phys. Chem. B 2019, 123 (13), 2756-2769] to assign structures of protein complexes in the range from â¼16 to â¼60 kDa from their "native" IM/MS spectra. Our analysis shows that the computed IM/MS spectra agree with the experimental spectra within the errors of the methods. The structure relaxation approximation (SRA) indicates that native backbone contacts appear largely retained in the absence of solvent for the investigated protein complexes and charge states. Native contacts between polypeptide chains of the protein complex appear to be retained to a comparable extent as contacts within a folded polypeptide chain. Our computations also indicate that the hallmark "compaction" often observed for protein systems in native IM/MS measurements appears to be a poor indicator of the extent to which native residue-residue interactions are lost in the absence of solvent. Further, the SRA indicates that structural reorganization of the protein systems in IM/MS measurements appears driven largely by remodeling of the protein surface that increases its hydrophobic content by approximately 10%. For the systems studied here, this remodeling of the protein surface appears to occur mainly by structural reorganization of surface-associated hydrophilic amino acid residues not associated with ß-strand secondary structure elements. Properties related to the internal protein structure, as assessed by void volume or packing density, appear unaffected by remodeling of the surface. Taken together, the structural reorganization of the protein surface appears to be generic in nature and to sufficiently stabilize protein structures to render them metastable on the time scale of IM/MS measurements.
Assuntos
Proteínas de Membrana , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização por Electrospray/métodos , Aminoácidos , Íons/química , SolventesRESUMO
"Top-down" proteomics analyzes intact proteins and identifies proteoforms by their intact mass as well as the observed fragmentation pattern in tandem mass spectrometry (MS/MS) experiments. Recently, hybrid ion mobility spectrometry-mass spectrometry (IM/MS) methods have gained traction for top-down experiments, either by allowing top-down analysis of individual isomers or alternatively by improving signal/noise and dynamic range for fragment ion assignment. We recently described the construction of a tandem-trapped ion mobility spectrometer/mass spectrometer (tandem-TIMS/MS) coupled with an ultraviolet (UV) laser and demonstrated a proof-of-principle for top-down analysis by UV photodissociation (UVPD) at 2-3 mbar. The present work builds on this with an exploration of a top-down method that couples tandem-TIMS/MS with UVPD and parallel-accumulation serial fragmentation (PASEF) MS/MS analysis. We first survey types and structures of UVPD-specific fragment ions generated in the 2-3 mbar pressure regime of our instrument. Notably, we observe UVPD-induced fragment ions with multiple conformations that differ from those produced in the absence of UV irradiation. Subsequently, we discuss how MS/MS spectra of top-down fragment ions lend themselves ideally for probability-based scoring methods developed in the bottom-up proteomics field and how the ability to record automated PASEF-MS/MS spectra resolves ambiguities in the assignment of top-down fragment ions. Finally, we describe the coupling of tandem-TIMS/MS workflows with UVPD and PASEF-MS/MS analysis for native top-down protein analysis.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Proteínas/análise , Íons , Raios UltravioletaRESUMO
Ion mobility spectrometry-mass spectrometry methods offer the potential to correlate protein tertiary and quaternary structures to variations in their amino acid sequences and post-translational modifications. Because ion mobility spectrometry measures cross sections of ions in the gas phase, however, the structure of protein systems detected by ion mobility spectrometry will generally differ from their native solution structures. While it is now established that ion mobility spectrometry does not typically detect equilibrium gas-phase structures of protein systems, what remains disputed is which aspects, if any, of the detected ions resemble the native state present in solution. Here, we develop the structure relaxation approximation (SRA) method to predict charge-state specific ion mobility spectra from an ensemble of solution structures. This allows us to predict the "global" trends observed in the experiments for various experimental conditions and charge states, thereby enabling detailed structure elucidation. The SRA predicts (RMSD to experiment â¼4%) that even the small protein ubiquitin largely retains its native inter-residue contacts with an intact hydrophobic core when studied by "soft" ion mobility measurements. Because collisional activation is increasingly inefficient with increasing numbers of internal degrees of freedom, the SRA suggests that it is all the more likely that ion mobility spectrometry retains essentially the native state for protein systems larger than ubiquitin.
Assuntos
Espectrometria de Mobilidade Iônica , Proteínas/química , Modelos Moleculares , Conformação ProteicaRESUMO
Ion mobility spectrometry-mass spectrometry (IMS-MS) has demonstrated the ability to characterize structures of weakly-bound peptide assemblies. However, these assemblies can potentially dissociate during the IMS-MS measurement if they undergo energetic ion-neutral collisions. Here, we investigate the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (TIMS-TIMS-MS) to retain weakly-bound peptide assemblies. We assess ion heating and dissociaton in the tandem-TIMS instrument using bradykinin and its assemblies as reference systems. Our data indicate that non-covalent bradykinin assemblies are largely preserved in TIMS-TIMS under carefully selected operating conditions. Importantly, we observe quadruply-charged bradykinin tetramers, which attests to the "softness" of our instrument. Graphical Abstract.
Assuntos
Bradicinina/química , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas em Tandem/métodos , Desenho de Equipamento , Calefação , Espectrometria de Mobilidade Iônica/instrumentação , Íons/química , Multimerização Proteica , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
: In this chapter, we discuss the state-of-the-art peptide array technologies, comparing the spot technique, lithographical methods, and microelectronic chip-based approaches. Based on this analysis, we describe a novel peptide array synthesis method with a microelectronic chip printer. By means of a complementary metal oxide semiconductor chip, charged bioparticles can be patterned on its surface. The bioparticles serve as vehicles to transfer molecule monomers to specific synthesis spots. Our chip offers 16,384 pixel electrodes on its surface with a spot-to-spot pitch of 100 µm. By switching the voltage of each pixel between 0 and 100 V separately, it is possible to generate arbitrary particle patterns for combinatorial molecule synthesis. Afterwards, the patterned chip surface serves as a printing head to transfer the particle pattern from its surface to a synthesis substrate. We conducted a series of proof-of-principle experiments to synthesize high-density peptide arrays. Our solid phase synthesis approach is based on the 9-fluorenylmethoxycarbonyl protection group strategy. After melting the particles, embedded monomers diffuse to the surface and participate in the coupling reaction to the surface. The method demonstrated herein can be easily extended to the synthesis of more complicated artificial molecules by using bioparticles with artificial molecular building blocks. The possibility of synthesizing artificial peptides was also shown in an experiment in which we patterned biotin particles in a high-density array format. These results open the road to the development of peptide-based functional modules for diverse applications in biotechnology.