High-Throughput Mass Spectrometry for Biopharma: A Universal Modality and Target Independent Analytical Method for Accurate Biomolecule Characterization.

Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size.

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides.

A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1.

Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein.

A cocktail of thermally stable, chemically synthesized capture agents for the efficient detection of anti-gp41 antibodies from human sera.

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen.

Femtosecond laser-assisted optoporation for drug and gene delivery into single mammalian cells.

In this work, focused near-infrared (NIR) femtosecond laser pulses were used to transiently perforate the cellular membrane of targeted human embryonic kidney (HEK) cells and the uptake of extrinsic molecules into the targeted cells was observed. Various cellular responses to the laser treatments were closely analyzed to optimize several experimental parameters such as laser power, exposure time and location of laser irradiation using a membrane impermeable fluorescent dye. The optimized parameters were used to investigate the entry of a plasmid DNA encoding green fluorescent protein (GFP) into the target cells.

Transforming a pair of orthogonal tRNA-aminoacyl-tRNA synthetase from Archaea to function in mammalian cells.

A previously engineered Methanocaldococcus jannaschii tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells.

A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker.

The potential usefulness of artificially selected peptides as probes to detect specific proteins has been proposed because of the ease and low cost of syntheses, manipulation, and genetic expression. However, the affinities of these peptides to their target proteins are generally too low to be practical as diagnostic or bioanalytical reagents. One approach to this problem is to incorporate a redox-active amino acid, 3,4-dihydroxy-l-phenylalanine (l-DOPA), that selectively forms a covalent linkage to the target protein.

Site-specific protein cross-linking with genetically incorporated 3,4-dihydroxy-L-phenylalanine.

Come together right now with L-DOPA: Chemical cross-linking is widely used to study protein-protein interactions. However, many cross-linking agents suffer from low reactivity or selectivity. An efficient and selective reaction of site-specific protein cross-linking was achieved using genetically incorporated 3,4-dihydroxy-L-phenylalanine.

A tetracycline repressor-based mammalian two-hybrid system to detect protein-protein interactions in vivo.

A mammalian two-hybrid system (termed as trM2H) was developed to detect protein-protein interactions in vivo, based on the reconstitution of the functions the of tetracycline repressor (TetR). The system is sensitive enough to detect protein-protein interactions with K(d) up to 55microM in mammalian cells, and the system can be regulated by small molecules. This system can be used as an efficient genetic selection system to map protein-protein interactions in mammalian cells.

Single mutation on the surface of Staphylococcus aureus Sortase A can disrupt its dimerization.

Staphylococcus aureus Sortase A (SrtA) is an important Gram-positive membrane enzyme which catalyzes the anchoring of many cell surface proteins conserved with the LPXTG sequence. Recently SrtA has been demonstrated to be a dimer with a Kd of 55 microM in vitro. Herein, we show that a single point mutation of amino acid residue on the surface of SrtA can completely disrupt the dimerization.

Staphylococcus aureus sortase A exists as a dimeric protein in vitro.

We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein.