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.

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.

Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from blood remains an unsolved challenge due to the extraordinary dynamic range of the blood proteome. Here, we develop an integrated nanoproteomics method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of cardiac troponin I (cTnI), a gold-standard cardiac biomarker, directly from serum. These NPs enable the sensitive enrichment of cTnI (<1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant proteins such as human serum albumin (>10 10 more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide highresolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibodyfree strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.

Metabolomicsthe endpoint of the omics cascadeis increasingly recognized as a preferred method for understanding the ultimate responses of biological systems to stress. Flow injection electrospray (FIE) mass spectrometry (MS) has advantages for untargeted metabolic fingerprinting due to its simplicity and capability for high-throughput screening but requires a high-resolution mass spectrometer to resolve metabolite features. In this study, we developed and validated a high-throughput and highly reproducible metabolomics platform integrating FIE with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS for analysis of both polar and nonpolar metabolite features from plasma samples. FIE-FTICR MS enables high-throughput detection of hundreds of metabolite features in a single mass spectrum without a front-end separation step. Using plasma samples from genetically identical obese mice with or without type 2 diabetes (T2D), we validated the intra and intersample reproducibility of our method and its robustness for simultaneously detecting alterations in both polar and nonpolar metabolite features. Only 5 min is needed to acquire an ultra-high resolution mass spectrum in either a positive or negative ionization mode. Approximately 1000 metabolic features were reproducibly detected and annotated in each mouse plasma group. For significantly altered and highly abundant metabolite features, targeted tandem MS (MS/MS) analyses can be applied to confirm their identity. With this integrated platform, we successfully detected over 300 statistically significant metabolic features in T2D mouse plasma as compared to controls and identified new T2D biomarker candidates. This FIE-FTICR MS-based method is of high throughput and highly reproducible with great promise for metabolomics studies toward a better understanding and diagnosis of human diseases.

A reproducible synthetic strategy was developed for facile large-scale (200 mg) synthesis of surface silanized magnetite (Fe3O4) nanoparticles (NPs) for biological applications. After further coupling a phosphate-specific affinity ligand, these functionalized magnetic NPs were used for the highly specific enrichment of phosphoproteins from a complex biological mixture. Moreover, correlating the surface silane density of the silanized magnetite NPs to their resultant enrichment performance established a simple and reliable quality assurance control to ensure reproducible synthesis of these NPs routinely in large scale and optimal phosphoprotein enrichment performance from batch-to-batch. Furthermore, by successful exploitation of a top-down phosphoproteomics strategy that integrates this high throughput nanoproteomics platform with online liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we were able to specifically enrich, identify, and characterize endogenous phosphoproteins from highly complex human cardiac tissue homogenate. This nanoproteomics platform possesses a unique combination of scalability, specificity, reproducibility, and efficiency for the capture and enrichment of low abundance proteins in general, thereby enabling downstream proteomics applications.

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).

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