Effect of buffer cations and of H3O+ on the charge states of native proteins. Significance to determinations of stability constants of protein complexes

Complement damages membranes via the terminal reaction sequence that leads to the formation of membrane-bound, macromolecular C5b-9(m) protein complexes. These complexes represent C5b-8 monomers to which varying numbers of C9 molecules can be bound. Complexes carrying high numbers of C9 ( ca . 6/8-12/16?) exhibit the morphology of hollow protein channels. Because they are embedded within the lipid bilayer, aqueous transmembrane pores are generated that represent the primary lesions caused by complement in the target cell membrane. Many other proteins damage membranes by forming channels in a manner analogous to the C5b-9(m) complex. Two prototypes of bacterial exotoxins, Staphylococcus aureus α-toxin and streptolysin-O, are discussed in this context, and attention is drawn to the numerous analogies existing among these protein systems. Common to all is the process of self-association of the native proteins to form supramolecular complexes. This event is in turn accompanied by a unique transition of the molecules from a hydrophilic to an amphiphilic state.

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Surface-Induced Dissociation of Anionic vs Cationic Native-like Protein Complexes

10.26434/chemrxiv.13547837.v2 ◽

2021 ◽

Author(s):

Sophie Harvey ◽

Zachary VanAernum ◽

Vicki Wysocki

Keyword(s):

Protein Interactions ◽

Structural Information ◽

Protein Complexes ◽

Charge Reduction ◽

Surface Induced Dissociation ◽

Negative Mode ◽

Peak Broadening ◽

Lower Charge ◽

Positive Mode ◽

Charge States

Characterizing protein-protein interactions, stoichiometries, and subunit connectivity is key to understanding how subunits assemble in biologically relevant multi-subunit protein complexes. Native mass spectrometry (nMS) has emerged as a powerful tool to study protein complexes due to its low sample requirements and tolerance for heterogeneity. For such nMS studies, positive mode ionization is routinely used and charge reduction, through the addition of solution additives, is often used, as the resulting lower charge states are often more compact and considered more native like. When studied with surface-induced dissociation, charge reduced complexes often give increased structural information over their “normal-charged” counter parts. A disadvantage of charge-reduction is that increased adduction, and hence peak broadening, is often observed when charge-reducing solution additives are present. Recent studies have shown that protein complexes ionized using negative mode generally form in lower charge states relative to positive mode. Here we demonstrate that the lower charged protein complex anions, activated by SID in an ultrahigh mass range Orbitrap mass spectrometer, fragment in a manner consistent with their solved structure, hence providing substructural information. Negative mode ionization in ammonium acetate offers the advantage of charge reduction without the peak broadening associated with solution phase charge reduction additives and provides direct structural information, when coupled with SID.

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Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biosensors and Bioelectronics ◽

10.1016/s0956-5663(01)00229-9 ◽

2001 ◽

Vol 16 (9-12) ◽

pp. 1071-1078 ◽

Cited By ~ 37

Author(s):

Dobrin Nedelkov ◽

Randall W Nelson

Keyword(s):

Mass Spectrometry ◽

Limit Of Detection ◽

Interaction Analysis ◽

Protein Complexes ◽

Specific Binding ◽

Biological Fluids ◽

Biomolecular Interaction ◽

Biomolecular Interaction Analysis ◽

Native Proteins

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A study of ion-neutral collision cross-section values for low charge states of peptides, proteins, and peptide/protein complexes

International Journal of Mass Spectrometry ◽

10.1016/j.ijms.2009.10.009 ◽

2010 ◽

Vol 298 (1-3) ◽

pp. 111-118 ◽

Cited By ~ 35

Author(s):

Francisco A. Fernandez-Lima ◽

Ryan C. Blase ◽

David H. Russell

Keyword(s):

Cross Section ◽

Collision Cross Section ◽

Protein Complexes ◽

Charge States ◽

Low Charge

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Effects of Polarity on the Structures and Charge States of Native-Like Proteins and Protein Complexes in the Gas Phase

Analytical Chemistry ◽

10.1021/ac403139d ◽

2013 ◽

Vol 85 (24) ◽

pp. 12055-12061 ◽

Cited By ~ 58

Author(s):

Samuel J. Allen ◽

Alicia M. Schwartz ◽

Matthew F. Bush

Keyword(s):

Gas Phase ◽

Protein Complexes ◽

Charge States

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Characterization of IsdH (NEAT domain 3) and IsdB (NEAT domain 2) in Staphylococcus aureus by magnetic circular dichroism spectroscopy and electrospray ionization mass spectrometry

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424609001352 ◽

2009 ◽

Vol 13 (10) ◽

pp. 1006-1016 ◽

Cited By ~ 11

Author(s):

Michael T. Tiedemann ◽

Naomi Muryoi ◽

David E. Heinrichs ◽

Martin J. Stillman

Keyword(s):

Staphylococcus Aureus ◽

Electrospray Ionization ◽

Magnetic Circular Dichroism ◽

Ionization Mass ◽

Esi Ms ◽

Native Proteins ◽

Charge States ◽

Domain 3 ◽

Ferrous Heme ◽

Circular Dichroïsm

Absorption and magnetic circular dichroism (MCD) spectra, together with electrospray ionization mass spectral (ESI-MS) data are reported for the first two proteins in the Isd sequence of proteins in Staphylococcus aureus. IsdH-NEAT domain 3 (IsdH-N3) and IsdB-NEAT domain 2 (IsdB-N2) are considered to be involved in heme transport following heme scavenging from the hemoglobin of the host. The ESI-MS data show that a single heme binds to each of these NEAT domains. The charge states of the native proteins indicate that there is minimal change in conformation when heme binds to the heme-free native protein. Acid denaturation releases the bound heme and results in protein that exhibits significantly higher charge states, which we associate with unfolding of the protein structure. MCD spectra of the heme-bound native proteins show that the heme-iron is in a high-spin state, which is similar to that in IsdC-N. Addition of cyanide results in a spectral envelope characteristic of low-spin ferric hemes. The lack of complete binding for IsdH-N3 suggests that there is considerable congestion in the heme-binding site region. Unusually, reduction to the ferrous heme results in spectral characteristics of six coordination of the ferrous heme. CO is shown to bind strongly to both heme bound proteins, resulting in six-coordinate bound hemes. The spectra following reduction most closely resemble spectra recorded for heme with histidine in the fifth position and methionine in the sixth position. We report a theoretical model calculated from the X-ray structure coordinates of IsdH-N3, in which the heme is coordinated to nearby histidine and methionine. We propose that this structure accounts for the spectroscopic properties of the protein with the ferrous heme.

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Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Analytical Chemistry ◽

10.1021/acs.analchem.6b02377 ◽

2017 ◽

Vol 89 (5) ◽

pp. 2731-2738 ◽

Cited By ~ 32

Author(s):

Huilin Li ◽

Yuewei Sheng ◽

William McGee ◽

Michael Cammarata ◽

Dustin Holden ◽

...

Keyword(s):

Mass Spectrometry ◽

Structural Characterization ◽

Protein Complexes ◽

Electron Ionization ◽

Native Proteins

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Complexome Profiling—Exploring Mitochondrial Protein Complexes in Health and Disease

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.796128 ◽

2022 ◽

Vol 9 ◽

Author(s):

Alfredo Cabrera-Orefice ◽

Alisa Potter ◽

Felix Evers ◽

Johannes F. Hevler ◽

Sergio Guerrero-Castillo

Keyword(s):

Protein Complexes ◽

Gradient Centrifugation ◽

Mitochondrial Protein ◽

Size Exclusion ◽

Native Proteins ◽

Mass Spectrometry Identification ◽

Exclusion Chromatography ◽

Health And Disease ◽

The Impact ◽

Novel Protein

Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions. Besides, complexome profiling has been utilized to study the dynamics of OXPHOS complexes, as well as the impact of an increasing number of mutations leading to mitochondrial disorders or rearrangements of the whole mitochondrial complexome. Here, we summarize the major findings obtained by this approach; emphasize its advantages and current limitations; discuss multiple examples on how this tool could be applied to further investigate pathophysiological mechanisms and comment on the latest advances and opportunity areas to keep developing this methodology.

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Surface-Induced Dissociation of Anionic vs Cationic Native-like Protein Complexes

10.26434/chemrxiv.13547837 ◽

2021 ◽

Author(s):

Sophie Harvey ◽

Zachary VanAernum ◽

Vicki Wysocki

Keyword(s):

Protein Interactions ◽

Structural Information ◽

Protein Complexes ◽

Charge Reduction ◽

Surface Induced Dissociation ◽

Negative Mode ◽

Peak Broadening ◽

Lower Charge ◽

Positive Mode ◽

Charge States

Characterizing protein-protein interactions, stoichiometries, and subunit connectivity is key to understanding how subunits assemble in biologically relevant multi-subunit protein complexes. Native mass spectrometry (nMS) has emerged as a powerful tool to study protein complexes due to its low sample requirements and tolerance for heterogeneity. For such nMS studies, positive mode ionization is routinely used and charge reduction, through the addition of solution additives, is often used, as the resulting lower charge states are often more compact and considered more native like. When studied with surface-induced dissociation, charge reduced complexes often give increased structural information over their “normal-charged” counter parts. A disadvantage of charge-reduction is that increased adduction, and hence peak broadening, is often observed when charge-reducing solution additives are present. Recent studies have shown that protein complexes ionized using negative mode generally form in lower charge states relative to positive mode. Here we demonstrate that the lower charged protein complex anions, activated by SID in an ultrahigh mass range Orbitrap mass spectrometer, fragment in a manner consistent with their solved structure, hence providing substructural information. Negative mode ionization in ammonium acetate offers the advantage of charge reduction without the peak broadening associated with solution phase charge reduction additives and provides direct structural information, when coupled with SID.

Download Full-text

Surface-Induced Dissociation of Anionic vs Cationic Native-like Protein Complexes

10.26434/chemrxiv.13547837.v1 ◽

2021 ◽

Author(s):

Sophie Harvey ◽

Zachary VanAernum ◽

Vicki Wysocki

Keyword(s):

Protein Interactions ◽

Structural Information ◽

Protein Complexes ◽

Charge Reduction ◽

Surface Induced Dissociation ◽

Negative Mode ◽

Peak Broadening ◽

Lower Charge ◽

Positive Mode ◽

Charge States

Characterizing protein-protein interactions, stoichiometries, and subunit connectivity is key to understanding how subunits assemble in biologically relevant multi-subunit protein complexes. Native mass spectrometry (nMS) has emerged as a powerful tool to study protein complexes due to its low sample requirements and tolerance for heterogeneity. For such nMS studies, positive mode ionization is routinely used and charge reduction, through the addition of solution additives, is often used, as the resulting lower charge states are often more compact and considered more native like. When studied with surface-induced dissociation, charge reduced complexes often give increased structural information over their “normal-charged” counter parts. A disadvantage of charge-reduction is that increased adduction, and hence peak broadening, is often observed when charge-reducing solution additives are present. Recent studies have shown that protein complexes ionized using negative mode generally form in lower charge states relative to positive mode. Here we demonstrate that the lower charged protein complex anions, activated by SID in an ultrahigh mass range Orbitrap mass spectrometer, fragment in a manner consistent with their solved structure, hence providing substructural information. Negative mode ionization in ammonium acetate offers the advantage of charge reduction without the peak broadening associated with solution phase charge reduction additives and provides direct structural information, when coupled with SID.

Download Full-text