27 research outputs found
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The Application of Hydrogen/Deuterium Exchange and Covalent Labeling Coupled with Mass Spectrometry to Examine Protein Structure
Thorough insight into a protein’s structure is necessary to understand how it functions and what goes wrong when it malfunctions. The structure of proteins, however, is not easily analyzed. The analysis must take place under a narrow range of conditions or risk perturbing the very structure being probed. Furthermore, the wide diversity in size and chemistry possible in proteins significantly complicates this analysis. Despite this numerous methods have been developed in order to analyze protein structure. In this work, we demonstrate that mass spectrometry (MS)-based techniques are capable of characterizing the structure of particularly challenging proteins. This is done through the study of two model systems: (1) the amyloid forming protein β2-microglobulin and (2) the protein therapeutics human growth hormone and immunoglobulin G1.
β-2-microglobulin (β2m) is an amyloidogenic protein and is the major constituent of fibrils in the disease dialysis related amyloidosis (DRA). Stoichiometric concentrations of Cu(II) have been used in vitro to induce the amyloid formation of β2m, but the structural changes caused by Cu(II) have not been fully elucidated. Other transition metals, such as Zn(II) and Ni(II), do not cause β2m amyloid formation, yet a comparison of the structural changes caused by these metals and those caused by Cu(II) could reveal essential structural changes necessary for amyloid formation. To probe these different structural changes, we have used a combination of hydrogen-deuterium exchange (HDX) and covalent labeling together with MS. Results from these measurements reveal that Cu(II) alone is capable of inducing the cis-trans isomerization of the X-Pro bond of Pro32 and the other necessary conformational changes that allow β2m to form an amyloid competent state, even though Ni(II) binds the protein at the same site. We also find that Zn(II) binding leads to increased dynamics, indicating increase structural instability, which is consistent with the amorphous aggregation observed in the presence of this metal.
The second part of this dissertation investigates the use of diethylpyrocarbonate (DEPC)–based covalent labeling to detect three-dimensional structural changes in immunoglobulin G1 and human growth hormone after they have been exposed to degrading conditions. We demonstrate that DEPC labeling can identify both specific protein regions that mediate aggregation and those regions that undergo more subtle structural changes upon mishandling of these proteins. Importantly, DEPC labeling is able to provide information for up to 30% of the surface residues in a given protein, thereby providing excellent structural resolution. Given the simplicity of the DEPC labeling chemistry and the relatively straightforward mass spectral analysis of DEPC-labeled proteins, we expect this method should be amenable to a wide range of protein therapeutics and their different formulations.
In the final section of this dissertation, we demonstrate that, in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low-charge state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification
Profiling Protein Sâ Sulfination with Maleimideâ Linked Probes
Cysteine residues are susceptible to oxidation to form Sâ sulfinyl (Râ SO2H) and Sâ sulfonyl (Râ SO3H) postâ translational modifications. Here we present a simple bioconjugation strategy to label Sâ sulfinated proteins by using reporterâ linked maleimides. After alkylation of free thiols with iodoacetamide, Sâ sulfinated cysteines react with maleimide to form a sulfone Michael adduct that remains stable under acidic conditions. Using this sequential alkylation strategy, we demonstrate differential Sâ sulfination across mouse tissue homogenates, as well as enhanced Sâ sulfination following pharmacological induction of endoplasmic reticulum stress, lipopolysaccharide stimulation, and inhibitors of the electron transport chain. Overall, this study reveals a broadened profile of maleimide reactivity across cysteine modifications, and outlines a simple method for profiling the physiological role of cysteine Sâ sulfination in disease.Maleimide, but not iodoacetamide, reacts with aryl and alkyl sulfinic acid standards and Sâ sulfinated proteins to give a sulfonylâ succinimide adduct that is stable under acidic conditions. This sequential alkylation strategy can be used for selective sulfinic acid labeling in biological samples. This study reveals a broadened profile of maleimide reactivity across cysteine modifications in proteins.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138861/1/cbic201700137_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138861/2/cbic201700137.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138861/3/cbic201700137-sup-0001-misc_information.pd
Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study
Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe
Identifying Zn-Bound Histidine Residues in Metalloproteins Using Hydrogen–Deuterium Exchange Mass Spectrometry
In
this work, we have developed a method that uses hydrogen–deuterium
exchange (HDX) of C2-hydrogens of histidines coupled with mass spectrometry
(MS) to identify Zn-bound histidines in metalloproteins. This method
relies on differences in HDX reaction rates of Zn-bound and Zn-free
His residues. Using several model peptides and proteins, we find that
all Zn-bound His residues have substantially lower HDX reaction rates
in the presence of the metal. The vast majority of non-Zn-binding
His residues undergo no significant changes in HDX reaction rates
when their reactivity is compared in the presence and absence of Zn.
Using this new approach, we then determined the Zn binding site of
β-2-microglobulin, a protein associated with metal-induced amyloidosis.
Together, these results suggest that HDX-MS of His C2-hydrogens is
a promising new method for identifying Zn-bound histidines in metalloproteins
Targeted Annotation of S‑Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry
Protein
S-sulfinylation (R–SO<sub>2</sub><sup>–</sup>) and S-sulfonylation
(R–SO<sub>3</sub><sup>–</sup>) are irreversible oxidative
post-translational modifications of
cysteine residues. Greater than 5% of cysteines are reported to occupy
these higher oxidation states, which effectively inactivate the corresponding
thiols and alter the electronic and physical properties of modified
proteins. Such higher oxidation states are reached after excessive
exposure to cellular oxidants, and accumulate across different disease
states. Despite widespread and functionally relevant cysteine oxidation
across the proteome, there are currently no robust methods to profile
higher order cysteine oxidation. Traditional data-dependent liquid
chromatography/tandem mass spectrometry (LC/MS/MS) methods generally
miss low-occupancy modifications in complex analyses. Here, we present
a data-independent acquisition (DIA) LC/MS-based approach, leveraging
the high IR absorbance of sulfoxides at 10.6 μm, for selective
dissociation and discovery of S-sulfonated peptides. Across peptide
standards and protein digests, we demonstrate selective infrared multiphoton
dissociation (IRMPD) of S-sulfonated peptides in the background of
unmodified peptides. This selective DIA IRMPD LC/MS-based approach
allows identification and annotation of S-sulfonated peptides across
complex mixtures while providing sufficient sequence information to
localize the modification site
Increased β‑Sheet Dynamics and D–E Loop Repositioning Are Necessary for Cu(II)-Induced Amyloid Formation by β‑2-Microglobulin
β-2-Microglobulin
(β2m) forms amyloid fibrils in the
joints of patients undergoing dialysis treatment as a result of kidney
failure. One of the ways in which β2m can be induced to form
amyloid fibrils <i>in vitro</i> is via incubation with stoichiometric
amounts of Cu(II). To better understand the structural changes caused
by Cu(II) binding that allow β2m to form amyloid fibrils, we
compared the effect of Ni(II) and Zn(II) binding, which are two similarly
sized divalent metal ions that do not induce β2m amyloid formation.
Using hydrogen/deuterium exchange mass spectrometry (HDX/MS) and covalent
labeling MS, we find that Ni(II) has little effect on β2m structure,
despite binding in the same region of the protein as Cu(II). This
observation indicates that subtle differences in the organization
of residues around Cu(II) cause distant changes that are necessary
for oligomerization and eventual amyloid formation. One key difference
that we find is that only Cu(II), not Ni(II) or Zn(II), is able to
cause the <i>cis</i>–<i>trans</i> isomerization
of Pro32 that is an important conformational switch that initiates
β2m amyloid formation. By comparing HDX/MS data from the three
metal-β2m complexes, we also discover that increased dynamics
in the β-sheet formed by the A, B, D, and E β strands
of the protein and repositioning of residues in the D–E loop
are necessary aspects of β2m forming an amyloid-competent dimer.
Altogether, our results reveal new structural insights into the unique
effect of Cu(II) in the metal-induced amyloid formation of β2m
Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry
Tandem
mass spectrometry (MS/MS) is the primary method for discovering,
identifying, and localizing post-translational modifications (PTMs)
in proteins. However, conventional positive ion mode collision induced
dissociation (CID)-based MS/MS often fails to yield site-specific
information for labile and acidic modifications due to low ionization
efficiency in positive ion mode and/or preferential PTM loss. While
a number of alternative methods have been developed to address this
issue, most require specialized instrumentation or indirect detection.
In this work, we present an amine-reactive TEMPO-based free radical
initiated peptide sequencing (FRIPS) approach for negative ion mode
analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation
generates sequence informative ions for both phosphorylated and sulfated
peptides with no significant PTM loss. Furthermore, FRIPS is compared
to positive ion mode CID, electron transfer dissociation (ETD), as
well as negative ion mode electron capture dissociation (niECD) and
CID, both in terms of sequence coverage and fragmentation efficiency
for phospho- and sulfo-peptides. Because FRIPS-based fragmentation
has no particular instrumentation requirements and shows limited PTM
loss, we propose this approach as a promising alternative to current
techniques for analysis of labile and acidic PTMs
Unique Effect of Cu(II) in the Metal-Induced Amyloid Formation of β‑2-Microglobulin
β-2-Microglobulin
(β2m) forms amyloid fibrils in the
joints of patients undergoing hemodialysis treatment as a result of
kidney failure. In the presence of stoichiometric amounts of Cu(II),
β2m self-associates into discrete oligomeric species, including
dimers, tetramers, and hexamers, before ultimately forming amyloid
fibrils that contain no copper. To improve our understanding of whether
Cu(II) is unique in its ability to induce β2m amyloid formation
and to delineate the coordinative interactions that allow Cu(II) to
exert its effect, we have examined the binding of Ni(II) and Zn(II)
to β2m and the resulting influence that these metals have on
β2m aggregation. We find that, in contrast to Cu(II), Ni(II)
does not induce the oligomerization or aggregation of β2m, while
Zn(II) promotes oligomerization but not amyloid fibril formation.
Using X-ray absorption spectroscopy and new mass spectrometry-related
techniques, we find that different binding modes are responsible for
the different effects of Ni(II) and Zn(II). By comparing the binding
modes of Cu(II) with Ni(II), we find that Cu(II) binding to Asp59
and the backbone amide between the first two residues of β2m
are important for allowing the formation of amyloid-competent oligomers,
as Ni(II) appears not to bind these sites on the protein. The oligomers
formed in the presence of Zn(II) are permitted by this metal’s
ability to bridge two β2m units via His51. These oligomers,
however, are not able to progress to form amyloid fibrils because
Zn(II) does not induce the required structural changes near the N-terminus
and His31
Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry
Protein therapeutics are rapidly
transforming the pharmaceutical
industry. Unlike for small molecule therapeutics, current technologies
are challenged to provide the rapid, high-resolution analyses of protein
higher order structures needed to ensure drug efficacy and safety.
Consequently, significant attention has turned to developing new methods
that can quickly, accurately, and reproducibly characterize the three-dimensional
structure of protein therapeutics. In this work, we describe a method
that uses diethylpyrocarbonate (DEPC) labeling and mass spectrometry
to detect three-dimensional structural changes in therapeutic proteins
that have been exposed to degrading conditions. Using β2-microglobulin,
immunoglobulin G1, and human growth hormone as model systems, we demonstrate
that DEPC labeling can identify both specific protein regions that
mediate aggregation and those regions that undergo more subtle structural
changes upon mishandling of these proteins. Importantly, DEPC labeling
is able to provide information for up to 30% of the surface residues
in a given protein, thereby providing excellent structural resolution.
Given the simplicity of the DEPC labeling chemistry and the relatively
straightforward mass spectral analysis of DEPC-labeled proteins, we
expect this method should be amenable to a wide range of protein therapeutics
and their different formulations
Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and ΔN6 β₂-Microglobulin
Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochemical oxidation of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β₂-microglobulin (β₂m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimised LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures