Native Mass Spectrometry Gives Insight into the Allosteric Binding Mechanism of M2 Pyruvate Kinase to Fructose-1,6-Bisphosphate

AbstractThe eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

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Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016342 ◽

2020 ◽

pp. jbc.RA120.016342

Author(s):

Harry C Tjondro ◽

Julian Ugonotti ◽

Rebeca Kawahara ◽

Sayantani Chatterjee ◽

Ian Loke ◽

...

Keyword(s):

Mass Spectrometry ◽

Native Mass Spectrometry ◽

Oxidation Products ◽

Site Specific ◽

Functional Roles ◽

Glycosylation Sites ◽

Molecular Complexity ◽

Functional Relevance ◽

Relationship Of ◽

Insight Into

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

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Insight into the Autosomal-Dominant Inheritance Pattern of SOD1-Associated ALS from Native Mass Spectrometry

Journal of Molecular Biology ◽

10.1016/j.jmb.2020.09.025 ◽

2020 ◽

Vol 432 (23) ◽

pp. 5995-6002 ◽

Cited By ~ 1

Author(s):

Jelena Cveticanin ◽

Tridib Mondal ◽

Elizabeth M. Meiering ◽

Michal Sharon ◽

Amnon Horovitz

Keyword(s):

Mass Spectrometry ◽

Autosomal Dominant ◽

Native Mass Spectrometry ◽

Autosomal Dominant Inheritance ◽

Inheritance Pattern ◽

Dominant Inheritance ◽

Insight Into ◽

Autosomal Dominant Inheritance Pattern

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Utilizing Ion Mobility-Mass Spectrometry to Investigate the Unfolding Pathway of Cu/Zn Superoxide Dismutase

Frontiers in Chemistry ◽

10.3389/fchem.2021.614595 ◽

2021 ◽

Vol 9 ◽

Author(s):

Karen E. Butler ◽

Yoshihiko Takinami ◽

Adam Rainczuk ◽

Erin S. Baker ◽

Blaine R. Roberts

Keyword(s):

Mass Spectrometry ◽

Superoxide Dismutase ◽

Protein Interactions ◽

Ion Mobility ◽

Quaternary Structure ◽

Structural Information ◽

Collision Cross Section ◽

Native Mass Spectrometry ◽

Structure Of Proteins ◽

Insight Into

Native mass spectrometry has emerged as a powerful tool for structural biology as it enables the evaluation of molecules as they occur in their physiological conditions. Ion mobility spectrometry-mass spectrometry (IMS-MS) has shown essential in these analyses as it allows the measurement of the shape of a molecule, denoted as its collision cross section (CCS), and mass. The structural information garnered from native IMS-MS provides insight into the tertiary and quaternary structure of proteins and can be used to validate NMR or crystallographic X-ray structures. Additionally, due to the rapid nature (millisecond measurements) and ability of IMS-MS to analyze heterogeneous solutions, it can be used to address structural questions not possible with traditional structural approaches. Herein, we applied multiple solution conditions to systematically denature bovine Cu/Zn-superoxide dismutase (SOD1) and assess its unfolding pathway from the holo-dimer to the holo-monomer, single-metal monomer, and apo-monomer. Additionally, we compared and noted 1–2% agreement between CCS values from both drift tube IMS and trapped IMS for the SOD1 holo-monomer and holo-dimer. The observed CCS values were in excellent agreement with computational CCS values predicted from the homo-dimer crystal structure, showcasing the ability to use both IMS-MS platforms to provide valuable structural information for molecular modeling of protein interactions and structural assessments.

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Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

10.1101/2020.07.24.219956 ◽

2020 ◽

Author(s):

Harry C. Tjondro ◽

Julian Ugonotti ◽

Rebeca Kawahara ◽

Sayantani Chatterjee ◽

Ian Loke ◽

...

Keyword(s):

Mass Spectrometry ◽

Native Mass Spectrometry ◽

Oxidation Products ◽

Multifaceted Approach ◽

Functional Roles ◽

Glycosylation Sites ◽

Functional Relevance ◽

Relationship Of ◽

First Time ◽

Insight Into

AbstractMyeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, structural modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.SignificanceMyeloperoxidase (MPO) is an important microbicidal glycoprotein critical for fighting pathogens. We report, for the first time, the intriguingly complex relationship between glycobiology and MPO immune function by demonstrating that uncommon and strategically positioned hyper-truncated glycans both elevate the activity and the inhibition potential of this pathogen-combating enzyme. We have used a multifaceted approach employing integrated biomolecular analytics to generate new insights into the sugar code of MPO. The findings described in this study improve our understanding of key innate immune processes and may guide future glycoengineering efforts aiming to generate therapeutically relevant recombinant MPO products with tuneable activity and inhibition potential tailored to biomedical applications involving persisting and severe pathogen infections.

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Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

10.26434/chemrxiv.8792177 ◽

2019 ◽

Author(s):

Zachary VanAernum ◽

Florian Busch ◽

Benjamin J. Jones ◽

Mengxuan Jia ◽

Zibo Chen ◽

...

Keyword(s):

Mass Spectrometry ◽

High Speed ◽

Structural Information ◽

Protein Complexes ◽

High Sensitivity ◽

Native Mass Spectrometry ◽

Structural Features ◽

Consumer Products ◽

Cell Lysates ◽

Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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