On the Preservation of Non-covalent Peptide Assemblies in a Tandem-Trapped Ion Mobility Spectrometer-Mass Spectrometer (TIMS-TIMS-MS)

Kirk, Samuel R.; Liu, Fanny C.; Cropley, Tyler C.; Carlock, Hunter R.; Bleiholder, Christian

doi:10.1007/s13361-019-02200-y

Cited by 32 publications

(69 citation statements)

References 57 publications

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“…TIMS is a relatively recent high-resolution ion mobility method in which multiple ions are held in place against a moving gas under an electric field and then ejected at characteristic elution voltages from the TIMS cell enabling the determination of ion mobility (1/ K 0 ) for structural analysis. 53 − 55 The mobility measured in TIMS allows collision cross-section (CCS) determination of protein conformers, which relates to their overall size and shape, and consequently can be used to evaluate changes in the three-dimensional structure. 54 , 57 However, ion mobility mapping and MS 1 analysis reveal enormous complexity in the native S-RBD sample ( Figure S2 ).…”

Section: Resultsmentioning

confidence: 99%

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Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

et al. 2021

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry, and the S protein glycosylation plays key roles in altering the viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants as well as other O-glycoproteins in general.

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Section: Resultsmentioning

confidence: 99%

“…Equation 1 was selected to agree with previously published CCS calculations. 54 , 55 , 70 Theoretical CCS values were determined using the IMPACT method. 59 …”

Section: Methodsmentioning

confidence: 99%

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

et al. 2021

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“…Equation 1 was selected to agree with previously published CCS calculations. 57,58,68 Theoretical CCS were calculated using the IMPACT method. 60…”

Section: Sample Preparationmentioning

confidence: 99%

“…Here we developed a method for the molecularly detailed analysis of intact O-glycan proteoforms of the S-RBD by top-down MS (Figure 1). For the first time, we harness the capabilities of a hybrid TIMS quadrupole time-of-flight (QTOF) mass spectrometer (timsTOF Pro) (Figure 1C), which provides high resolving power and sensitivity for both selective and comprehensive ion mobility separations of various protein structural variants, [56][57][58][59] and the ultrahigh-resolution of a 12T Fourier Transform Ion Cyclotron Resonance (FTICR) MS (Figure 1D), to comprehensively characterize the O-glycoforms of the S-RBD, including the exact glycan structures and relative molecular abundance (Figure 1E). We demonstrate that this native top-down MS approach can provide a high-resolution and wholistic proteoform-resolved landscape of diverse O-glycoforms to enable future structure-function studies of the S-RBD.…”

Section: Introductionmentioning

confidence: 99%

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Roberts

Mann

Melby

et al. 2021

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.

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“…Ions of different mobilities are trapped at different points (potentials) along the ion optical axis using the electric field gradient and are then eluted from the device over time, based on their mobility, as the TIMS potential gradient is reduced. [18][19][20][21][22][23] TIMS, or an alternative IM approach called FAIMS, are often coupled with liquid chromatography in "omics" based mass spectrometry experiments, such as proteomics and metabolomics, in which complex biological samples require orthogonal modes of separation to separate and identify the numerous, structurally similar components. [18][19][20][24][25] In addition to TIMS serving as a separation technique, the collision cross section (CCS), a rotationally averaged measurement of an ion's structure, can be determined from the measured mobility value, thus providing insight into protein shape and conformation.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a Bruker timsTOF Pro for Native Mass Spectrometry

Panczyk

Snyder

Liu

et al. 2021

Preprint

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Mass spectrometry-based assays in structural biology studies typically measure either intact or digested proteins. Traditionally, there are different mass spectrometers dedicated for such measurements: those focused on the rapid analysis of small molecules, such as digested peptides, and those designed for high mass intact analysis. The Bruker timsTOF Pro mass spectrometer (ion mobility-quadrupole-time of flight platform, IM-Q-TOF), has become widely utilized for the analysis of small molecules in the fields of proteomics and metabolomics, with ion mobility spectrometry offering an additional stage of ion separation coupled to liquid chromatography. While this instrument has proven capabilities for small molecule analysis, the ability to perform high-quality native mass spectrometry of intact protein complexes remains largely uninvestigated. Here, we evaluate this IM-Q-TOF platform for the analysis of intact proteins and non-covalently bound protein complexes as small as 12 kDa (cytochrome c) and as large as 801 kDa (GroEL), utilizing the full range of ion mobility, MS, and MS/MS (in-source cleanup and collision cell CID) experiments available on this platform. In-source activation and collision cell CID were found to be robust capabilities for both small and large complexes. Nonetheless, the TIMS analyzer was soft enough to preserve protein-ligand interactions between 1,3-benzenedisulfonamide and carbonic anhydrase. TIMS-CID was performed on the protein complexes streptavidin (53 kDa), avidin (68 kDa) and cholera toxin B (CTB, 58 kDa). Pyruvate kinase and GroEL were beyond the trapping capabilities of the commercial TIMS analyzer. Although quadrupole selection is limited to m/z 3000 by the manufacturer, ions of significantly higher m/z can be transmitted and studied. The present results show that the commercially available Bruker IM-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for the TIMS analyzer and quadrupole will be required if protein complexes greater than a few tens of kDa are of interest.

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On the Preservation of Non-covalent Peptide Assemblies in a Tandem-Trapped Ion Mobility Spectrometer-Mass Spectrometer (TIMS-TIMS-MS)

Cited by 32 publications

References 57 publications

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

Evaluation of a Bruker timsTOF Pro for Native Mass Spectrometry

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