Biology subjects

Byrd, E. J.

Publications and source records attributed to Byrd, E. J..

3 recordsLinked to original sources

bioRxiv2026

Cross-Platform Assessment of Sub-50 nm Nanopipette Emitters for Native Electrospray Ionization Mass Spectrometry

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nano-electrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across three mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, and tribrid-Orbitrap platforms) and a wide protein range (14.5-800 kDa). We analysed the intrinsically disordered protein alpha synuclein ( S; 14.5 kDa) and holo-myoglobin (17 kDa) over a range of concentrations (10 M-1 nM) and capillary voltages to determine limits of detection and define a gentle operating regime. We additionally observed reduced Na adduction and preservation of the Zn 2+-bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin (~44 kDa), malate dehydrogenase (~70 kDa), transferrin (80 kDa), glutamate dehydrogenase (~350 kDa), {beta}-galactosidase (~465 kDa), and GroEL (~800 kDa), were readily detected using nanopipette emitters. Compared with conventional 1-2 m internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of clarified bacterial lysate expressing -synuclein yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.

biochemistry↗

bioRxiv2025

Buffer-dependent conformational dynamics of α-synuclein revealed by nanopipette electrospray ionisation ion mobility mass spectrometry

Electrolyte conditions in vivo and in vitro are known to influence protein structure and function. Intrinsically disordered proteins (IDPs) are particularly sensitive to their solution conditions such as ionic strength and molecular crowding, and their dynamic structural ensembles rapidly respond to the cellular environment. While structural mass spectrometry (MS) techniques are uniquely able to capture aspects of this structural diversity, technical limitations have largely precluded the use of native MS approaches to interrogate the conformational rearrangements of IDPs in response to high concentrations of non-volatile salts. Here, we overcome this challenge by employing sub 100-nm nanopipette electrospray emitters for more gentle and salt-tolerant analysis to study the conformational distribution of -Synuclein (S) using native MS and ion mobility-MS in varied solution conditions, including in phosphate buffered saline. We show using native MS that it is possible to capture salt and buffer induced changes in the S conformational ensemble when using traditional biochemical buffers, which reflect structural changes from in silico predictions and in-solution measurements. This work demonstrates the power of nanopipette emitters for the study of IDPs, and establishes native MS as a method that can be routinely used to determine how solution conditions tune the conformational landscape of IDPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/671163v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@35ff69org.highwire.dtl.DTLVardef@113821aorg.highwire.dtl.DTLVardef@1c27b90org.highwire.dtl.DTLVardef@13e4db1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

The structural architecture of an α-synuclein toxic oligomer

Oligomeric species populated during -synuclein aggregation are considered key drivers of neurodegeneration in Parkinsons disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. PSM3 is a nanomolar peptide binder of -synuclein oligomers that inhibits aggregation by blocking oligomer to fibril conversion. Here, we investigate the binding of PSM3 to -synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSM3 selectively targets an -synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the monomeric and oligomeric states. This -synuclein region plays a pivotal role in oligomer to fibril conversion, as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early-onset Parkinsons disease, causes a conformational fluctuation in this region, leading to delayed oligomer to fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in -synuclein oligomers, advance our comprehension of oligomer to amyloid fibril conversion and reveal a new facet of -synuclein pathogenic mutations.

biophysics↗