5 research outputs found
Structure-Based Design of Dendritic Peptide Bolaamphiphiles for siRNA Delivery
Development
of safe and effective delivery vectors is a critical challenge for
the application of RNA interference (RNAi)-based biotechnologies.
In this study we show the rational design of a series of novel dendritic
peptide bolaamphiphile vectors that demonstrate high efficiency for
the delivery of small interfering RNA (siRNA) while exhibiting low
cytotoxicity and hemolytic activity. Systematic investigation into
structure–property relationships revealed an important correlation
between molecular design, self-assembled nanostructure, and biological
activity. The unique bolaamphiphile architecture proved a key factor
for improved complex stability and transfection efficiency. The optimal
vector contains a fluorocarbon core and exhibited enhanced delivery
efficiency to a variety of cell lines and improved serum resistance
when compared to hydrocarbon analogues and lipofectamine RNAiMAX.
In addition to introducing a promising new vector system for siRNA
delivery, the structure–property relationships and “fluorocarbon
effect” revealed herein offer critical insight for further
development of novel materials for nucleic acid delivery and other
biomaterial applications
Multifunctional Dendronized Peptide Polymer Platform for Safe and Effective siRNA Delivery
In
this study, we designed and synthesized a biodegradable dendronized
polypeptide (denpol) platform for delivery of small interfering RNA
(siRNA). The novel denpol architecture combines the multivalency of
dendrimers and conformational flexibility of linear polymers for optimal
siRNA binding. Multifunctional amino acids were incorporated onto
the dendrons and the structure was tuned both systematically and combinatorially
to select optimal vectors. By screening a focused library, we identified
several denpols that can effectively deliver siRNA to NIH 3T3 cells
in vitro and exhibit minimal toxicity. For comparison, the best-performing
denpol showed significantly improved transfection efficiency over
Lipofectamine in serum-containing media. Fluorescence intracellular
trafficking studies indicated that amphiphilicity is important for
cell uptake and that the buffering capacity of histidine facilitates
endosomal membrane rupture and therefore enhances the transfection
efficiency. The combination of high delivery efficiency in serum and
low cytotoxicity suggests the denpol system as a promising new carrier
for siRNA delivery
Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins
Protein phosphorylation
is a ubiquitous and critical post-translational
modification (PTM) involved in numerous cellular processes. Mass spectrometry
(MS)-based proteomics has emerged as the preferred technology for
protein identification, characterization, and quantification. Whereas
ionization/detection efficiency of peptides in electrospray ionization
(ESI)-MS are markedly influenced by the presence of phosphorylation,
the physicochemical properties of intact proteins are assumed not
to vary significantly due to the relatively smaller modification on
large intact proteins. Thus, the ionization/detection efficiency of
intact phosphoprotein is hypothesized not to alter appreciably for
subsequent MS quantification. However, this hypothesis has never been
rigorously tested. Herein, we systematically investigated the impact
of phosphorylation on ESI-MS quantification of mono- and multiply
phosphorylated proteins. We verified that a single phosphorylation
did not appreciably affect the ESI-MS quantification of phosphoproteins
as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation.
Moreover, different ionization and desolvation parameters did not
impact phosphoprotein quantification. In contrast to monophosphorylation,
multiphosphorylation noticeably affected ESI-MS quantification of
phosphoproteins likely due to differential ionization/detection efficiency
between unphosphorylated and phosphorylated proteoforms as shown in
the pentakis-phosphorylated β-casein (24 kDa)
Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum
Top-down proteomics can provide unique insights into the biological variations of protein biomarkers but detecting low-abundance proteins in body fluids remains challenging. Here, the authors develop a nanoparticle-based top-down proteomics approach enabling enrichment and detailed analysis of cardiac troponin I in human serum
Structure and dynamics of endogenous cardiac troponin complex in human heart tissue captured by native nanoproteomics
Abstract Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present challenges to study using conventional structural biology techniques. Here we develop a native nanoproteomics strategy for the enrichment and subsequent native top-down mass spectrometry (nTDMS) analysis of endogenous cardiac troponin (cTn) complex directly from human heart tissue. The cTn complex is enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complex, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the cTn complex, localizes Ca2+ binding domains, defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a paradigm for structural characterization of endogenous native protein complexes