scholarly journals Native Mass Spectrometry and Surface Induced Dissociation Provide Insight into the Post-Translational Modifications of Tetrameric AQP0 Isolated from Bovine Eye Lens

2022 ◽  
Author(s):  
Sophie R. Harvey ◽  
Carla O’Neale ◽  
Kevin L. Schey ◽  
Vicki H. Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Eye Lens ◽  
Post Translational Modifications ◽  
Surface Induced Dissociation ◽  
Insight Into

scholarly journals Expanding the structural analysis capabilities on an Orbitrap-based mass spectrometer for large macromolecular complexes

The Analyst ◽  
2018 ◽  
Vol 143 (1) ◽  
pp. 100-105 ◽  
Author(s):  
Kyle L. Fort ◽  
Michiel van de Waterbeemd ◽  
Dmitriy Boll ◽  
Maria Reinhardt-Szyba ◽  
Mikhail E. Belov ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Analysis ◽  
Mass Spectrometer ◽  
Biological Systems ◽  
Native Mass Spectrometry ◽  
Macromolecular Complexes ◽  
Insight Into

Native mass spectrometry can provide insight into the structure of macromolecular biological systems.

scholarly journals Probing the structure of nanodiscs using surface-induced dissociation mass spectrometry

2020 ◽  
Vol 56 (100) ◽  
pp. 15651-15654
Author(s):  
Sophie R. Harvey ◽  
Zachary L. VanAernum ◽  
Marius M. Kostelic ◽  
Michael T. Marty ◽  
Vicki H. Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Native Mass Spectrometry ◽  
Surface Induced Dissociation ◽  
Membrane Mimetic

Nanodiscs have emerged as a promising membrane mimetic, and have been utilized in native mass spectrometry studies. Here we use surface-induced dissociation to study the structure of nanodiscs in the gas-phase.

scholarly journals Mechanistic Insight into the Assembly of the HerA-NurA Helicase-Nuclease DNA End Resection Complex using Native Mass Spectrometry

2018 ◽  
Vol 114 (3) ◽  
pp. 440a
Author(s):  
Zainab Ahdash ◽  
Andy M. Lau ◽  
Robert Thomas Byrne ◽  
Katja Lammens ◽  
Paula J. Booth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Native Mass Spectrometry ◽  
Mechanistic Insight ◽  
Dna End Resection ◽  
End Resection ◽  
Insight Into

scholarly journals Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miranda P. Collier ◽  
Karen Betancourt Moreira ◽  
Kathy H. Li ◽  
Yu-Chan Chen ◽  
Daniel Itzhak ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Insect Cells ◽  
Native Mass Spectrometry ◽  
Cellular Protein ◽  
Small Population ◽  
Relative Stabilities ◽  
Assembly Mechanism ◽  
And Function ◽  
Insight Into

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.

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

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.

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

Biochemistry ◽  
2018 ◽  
Vol 57 (11) ◽  
pp. 1685-1689 ◽  
Author(s):  
Agni F. M. Gavriilidou ◽  
Finn P. Holding ◽  
Daniel Mayer ◽  
Joseph E. Coyle ◽  
Dmitry B. Veprintsev ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Pyruvate Kinase ◽  
Native Mass Spectrometry ◽  
Binding Mechanism ◽  
Fructose 1,6 Bisphosphate ◽  
Insight Into

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

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.

scholarly journals Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

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.

scholarly journals Measuring proteomes with long strings: A new, unconstrained paradigm in mass spectrum interpretation

2018 ◽  
Author(s):  
Arun Devabhaktuni ◽  
Niclas Olsson ◽  
Carlos Gonzales ◽  
Keith Rawson ◽  
Kavya Swaminathan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Long Strings ◽  
Search Method ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Search Spaces ◽  
Spectrum Interpretation ◽  
Proline Hydroxylation ◽  
Insight Into ◽  
Enrichment Methods

SummaryThousands of protein post-translational modifications (PTMs) dynamically impact nearly all cellular functions. Mass spectrometry is well suited to PTM identification, but proteome-scale analyses are biased towards PTMs with existing enrichment methods. To measure the full landscape of PTM regulation, software must overcome two fundamental challenges: intractably large search spaces and difficulty distinguishing correct from incorrect identifications. Here, we describe TagGraph, software that overcomes both challenges with a string-based search method orders of magnitude faster than current approaches, and probabilistic validation model optimized for PTM assignments. When applied to a human proteome map, TagGraph tripled confident identifications while revealing thousands of modification types on nearly one million sites spanning the proteome. We expand known sites by orders of magnitude for highly abundant yet understudied PTMs such as proline hydroxylation, and derive tissue-specific insight into these PTMs’ roles. TagGraph expands our ability to survey the full landscape of PTM function and regulation.

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