Polymeric nanoassemblies for enrichment and detection of peptides and proteins in human breast milk.

Human breast milk is an understudied biological fluid that may be useful for early detection of breast cancer. Methods for enriching and detecting biomarkers in human breast milk, however, are not as well-developed as compared with other biological fluids. In this work, we demonstrate a new enrichment method based on polymeric nanoassemblies that is capable of enhancing the mass spectrometry-based detection of peptides and proteins in human breast milk. In this method, positively charged nanoassemblies are used to selectively deplete abundant proteins in milk based on electrostatic interactions, which simplifies the mixture and enhances detection of positively charged peptides and proteins. Negatively charged nanoassemblies are used in a subsequent enrichment step to further enhance the detection and quantification of trace-level peptides and proteins. Together the depletion and enrichment steps allow model biomarkers to be detected at low nM levels, which are close to instrumental limits of detection. This new method not only demonstrates the ability to detect proteins in human breast milk but also provides an alternative approach for targeted protein detection in complex biological matrices. Graphical abstract.

Supramolecular Assemblies for Transporting Proteins Across an Immiscible Solvent Interface.

Polymeric supramolecular assemblies that can effectively transport proteins across an incompatible solvent interface are described. We show that electrostatics and ligand-protein interactions can be used to selectively transport proteins from an aqueous phase to organic phase. These transported proteins have been shown to maintain their tertiary structure and function. This approach opens up new possibilities for application of supramolecular assemblies in sensing, diagnostics and catalysis.

Molecular Features Influencing the Release of Peptides from Amphiphilic Polymeric Reverse Micelles.

Efficient and controlled release of peptides bound to polymeric reverse micelle assemblies can be achieved through the cooperative effects of disassembly and disruption of charge-charge interactions. Through the examination of various peptides and polymer architectures, we have identified the factors that affect the release efficiency of the electrostatically bound peptides. Peptide guests and polymers with a greater number of complementary charges result in less efficient release than peptides and polymers with lower numbers of charges. Interestingly, we find that the presence of adjacent charged groups on the monomeric unit of the polymer exhibits exceptionally low release efficiency, perhaps because of a chelate-like effect, even when the total polymer charge is lower. Overall, our findings inform the design principles for catch-and-release systems based on polymeric reverse micelles, which offer great versatility and tunability.

Improved mass spectrometric detection of acidic peptides by variations in the functional group pKa values of reverse micelle extraction agents.

Polymeric reverse micelles can be used to selectively extract peptides from complex mixtures via a two-phase extraction approach. In previous work, we have shown that the charge polarity of the hydrophilic functional group that is in the interior of the reverse micelle dictates the extraction selectivity. To investigate how the extraction is influenced by the inherent pKa of the functional group, we designed and tested a series of polymeric reverse micelles with variations in the hydrophilic functional group. From this series of polymers, we find that the extraction capability of the reverse micelles in an apolar phase is directly related to the aqueous phase pKa of the interior functional group, suggesting that the functional groups maintain their inherent chemistry even in the confined environment of the reverse micelle interior. Because these functional groups maintain their inherent pKa in the reverse micelle interior, they provide predictable extraction selectivity upon changes in aqueous phase pH. We exploit this finding to demonstrate that sulfonate-containing polymers can be used to remove basic peptides from complex mixtures, thereby allowing the improved detection of acidic peptides. Using these new materials, we also demonstrate a new means of isoelectric point (pI) bracketing that allows the mass spectrometric detection of peptides with a defined and narrow range of pI values.

Polymer-mediated ternary supramolecular interactions for sensitive detection of peptides.

A combination of donor-acceptor and electrostatic interactions in a three-component supramolecular system has been shown to form the basis for selective and sensitive detection of peptides. Different substituents in the polymer and the detection matrix were compared to demonstrate that the favorable donor-acceptor interactions explain the observed signal enhancement. The ternary supramolecular interactions discovered in this work are enabled by the self-packing behavior of amphiphilic homopolymers and their ability to mediate interactions between the detection matrix and peptide that facilitate sensitive detection of peptides.

Mass spectrometry in gene therapy: Challenges and opportunities for AAV analysis.

The characterization of adeno-associated virus (AAV)-based gene therapy products represents significant challenges owing to their extremely large molecular sizes, structural complexity and heterogeneity, and limited sample amounts. Mass spectrometry (MS) is one of the key analytical tools that can overcome these challenges and serve as an important technique for the analysis of multiple attributes. In this review, the current methodologies and emerging trends in MS analysis of AAV gene therapy products are presented, highlighting their advantages and unique capabilities in addressing key issues encountered in intact AAV vector analysis, capsid viral protein characterization and impurity analysis.

Supramolecular Polymeric Assemblies for the Selective Depletion of Abundant Acidic Proteins in Serum.

The analysis of low-level protein biomarkers in serum is precluded by the presence of other highly abundant serum proteins. Hence, the preliminary removal of serum albumin along with other abundant proteins in serum (i.e., immunoglobulins, transferrin, haptoglobin, α-2-macroglobulin, and apolipoproteins) is often a requirement prior to any biomarker analysis. In this work, we take advantage of the low isoelectric points (pI's) of these highly abundant proteins to selectively deplete them from serum by extraction using functionalized amphiphilic polymeric nanoassemblies. The selectivity of extraction is dependent on the pI of the protein and the extraction pH, which holds true even for extremely complex protein mixtures like serum. High extraction capacity is achieved by optimizing the extraction conditions and is found to be comparable to currently available methods for depletion. Depletion of these abundant acidic proteins allows for the enhanced detection of higher pI proteins and enables a 3 orders of magnitude increase in detection sensitivity for a putative cancer biomarker, demonstrating the utility of these polymeric assemblies for enhancing the analysis of the serum proteome.

Self-assembly of random co-polymers for selective binding and detection of peptides.

Amphiphilic random co-polymers, which form stable reverse micelle-type assemblies, have been designed and synthesized. We demonstrate that the reverse micelles, formed by these co-polymers are capable of selectively binding peptides through electrostatic interactions, indicating that these random polymers can self-organize into functionally selective materials. Moreover, these random co-polymers also enable the ordered co-crystallization of matrix and extracted guest molecules, giving rise to substantial signal enhancements during MALDI-MS detection. Together, these observations represent an excellent example of how random polymers can self-assemble into ordered, functional materials.