Using High-Throughput Experiments To Screen N-Glycosyltransferases with Altered Specificities.

The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X-1-N-X+1-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X+2 position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.

Tyrosine phosphatase activity is restricted by basic charge substituting mutation of substrates.

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two ß-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts ß-catenin from cell-cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant ß-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations.

Characterizing Enzyme Cooperativity with Imaging SAMDI-MS.

This paper describes a method that combines a microfluidic device and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 µL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca2+ . Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 µm pixel on the monolayer, providing a time course for the reactions. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K0.5 ) for the Ca2+ - dependent activation of PAD2. This work establishes a high-throughput and label-free method for studying enzyme-ligand binding interactions and widens the applicability of microfluidics and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry.

Morphological features of single cells enable accurate automated classification of cancer from non-cancer cell lines.

Accurate cancer detection and diagnosis is of utmost importance for reliable drug-response prediction. Successful cancer characterization relies on both genetic analysis and histological scans from tumor biopsies. It is known that the cytoskeleton is significantly altered in cancer, as cellular structure dynamically remodels to promote proliferation, migration, and metastasis. We exploited these structural differences with supervised feature extraction methods to introduce an algorithm that could distinguish cancer from non-cancer cells presented in high-resolution, single cell images. In this paper, we successfully identified the features with the most discriminatory power to successfully predict cell type with as few as 100 cells per cell line. This trait overcomes a key barrier of machine learning methodologies: insufficient data. Furthermore, normalizing cell shape via microcontact printing on self-assembled monolayers enabled better discrimination of cell lines with difficult-to-distinguish phenotypes. Classification accuracy remained robust as we tested dissimilar cell lines across various tissue origins, which supports the generalizability of our algorithm.

Exploring the Ligand Preferences of the PHD1 Domain of Histone Demethylase KDM5A Reveals Tolerance for Modifications of the Q5 Residue of Histone 3.

Understanding the ligand preferences of epigenetic reader domains enables identification of modification states of chromatin with which these domains associate and can yield insight into recruitment and catalysis of chromatin-acting complexes. However, thorough exploration of the ligand preferences of reader domains is hindered by the limitations of traditional protein-ligand binding assays. Here, we evaluate the binding preferences of the PHD1 domain of histone demethylase KDM5A using the protein interaction by SAMDI (PI-SAMDI) assay, which measures protein-ligand binding in a high-throughput and sensitive manner via binding-induced enhancement in the activity of a reporter enzyme, in combination with fluorescence polarization. The PI-SAMDI assay was validated by confirming its ability to accurately profile the relative binding affinity of a set of well-characterized histone 3 (H3) ligands of PHD1. The assay was then used to assess the affinity of PHD1 for 361 H3 mutant ligands, a select number of which were further characterized by fluorescence polarization. Together, these experiments revealed PHD1's tolerance for H3Q5 mutations, including an unexpected tolerance for aromatic residues in this position. Motivated by this finding, we further demonstrate a high-affinity interaction between PHD1 and recently identified Q5-serotonylated H3. This work yields interesting insights into permissible PHD1-H3 interactions and demonstrates the value of interfacing PI-SAMDI and fluorescence polarization in investigations of protein-ligand binding.

Synthetic Tuning of Domain Stoichiometry in Nanobody-Enzyme Megamolecules.

This paper presents a method to synthetically tune atomically precise megamolecule nanobody-enzyme conjugates for prodrug cancer therapy. Previous efforts to create heterobifunctional protein conjugates suffered from heterogeneity in domain stoichiometry, which in part led to the failure of antibody-enzyme conjugates in clinical trials. We used the megamolecule approach to synthesize anti-HER2 nanobody-cytosine deaminase conjugates with tunable numbers of nanobody and enzyme domains in a single, covalent molecule. Linking two nanobody domains to one enzyme domain improved avidity to a human cancer cell line by 4-fold but did not increase cytotoxicity significantly due to lowered enzyme activity. In contrast, a megamolecule composed of one nanobody and two enzyme domains resulted in an 8-fold improvement in the catalytic efficiency and increased the cytotoxic effect by over 5-fold in spheroid culture, indicating that the multimeric structure allowed for an increase in local drug activation. Our work demonstrates that the megamolecule strategy can be used to study structure-function relationships of protein conjugate therapeutics with synthetic control of protein domain stoichiometry.

Design and Synthesis of Megamolecule Mimics of a Therapeutic Antibody.

This communication describes the design, synthesis, and biological activity of a megamolecule mimic of an anti-HER2 antibody. The antibody mimic was prepared by linking two Fabs from the therapeutic antibody trastuzumab, which are fused through the heavy chain variable domain to either cutinase or SnapTag, with a linker terminated in an irreversible inhibitor for each enzyme. This mimic binds HER2 with comparable avidity to trastuzumab, has similar activity in a cell-based assay, and can arrest tumor growth in a mouse xenograft BT474 tumor model. A panel of 16 bivalent anti-HER2 antibodies were prepared wherein each varied in the orientation of the fusion domain on the Fabs. The analogs displayed a range of cytotoxic activity, and surprisingly, the most active mimic binds to cells with a 10-fold lower avidity than the least active variant suggesting that structure plays a large role in their efficacy. This work suggests that the megamolecule approach can be used to prepare antibody mimics having a broad structural diversity.

Outer-Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate.

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL-1 of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV ) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants.

Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry.

Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells.

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1.

The enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the conversion of isocitrate to alpha-ketoglutarate (αKG) and has emerged as an important therapeutic target for glioblastoma multiforme (GBM). Current methods for assaying IDH1 remain poorly suited for high-throughput screening of IDH1 antagonists. This paper describes a high-throughput and quantitative assay for IDH1 that is based on the self-assembled monolayers for matrix-assisted laser desorption/ionization-mass spectrometry (SAMDI-MS) method. The assay uses a self-assembled monolayer presenting a hydrazide group that covalently captures the αKG product of IDH1, where it can then be detected by MALDI-TOF mass spectrometry. Co-capture of an isotopically-labeled αKG internal standard allows the αKG concentration to be quantitated. The assay was used to analyze a series of standard αKG solutions and produced minimal error in measured αKG concentration values. The suitability of the assay for high-throughput analysis was evaluated in a 384-sample biochemical IDH1 screen. Cells expressing IDH1 were lysed and the lysate was applied to the monolayer to capture αKG, which was then quantitated using the SAMDI-MS assay. Cells in which IDH1 expression was reduced by small-interfering RNA exhibited a corresponding decrease in αKG concentration as measured by the assay. Application of the assay toward the high-throughput screening of IDH1 inhibitors or knockdown agents may facilitate the discovery of treatments for GBM.

Using Peptide Arrays to Profile Phosphatase Activity in Cell Lysates.

Phosphorylation is an important post-translational modification on proteins involved in many cellular processes; however, understanding of the regulation and mechanisms of global phosphorylation remains limited. Herein, we utilize self-assembled monolayers on gold for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) with three phosphorylated peptide arrays to profile global phosphatase activity in cell lysates derived from five mammalian cell lines. Our results reveal significant differences in the activities of protein phosphatases on phospho- serine, threonine, and tyrosine substrates and suggest that phosphatases play a much larger role in the regulation of global phosphorylation on proteins than previously understood.

Profiling Protein Tyrosine Phosphatase Specificity with Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Peptide Arrays.

The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This Research Article describes the use of phosphotyrosine-containing peptide arrays together with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to directly profile phosphatase substrate selectivities. Twenty-two protein tyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of a R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level and which this work suggests may be due to an increased activity toward phosphatase enzymes. The use of self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.

Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells.

Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation-during which the PTP enzymes act on the peptide substrate-the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.

High-throughput mapping of CoA metabolites by SAMDI-MS to optimize the cell-free biosynthesis of HMG-CoA.

Metabolic engineering uses enzymes to produce small molecules with industrial, pharmaceutical, and energy applications. However, efforts to optimize enzymatic pathways for commercial production are limited by the throughput of assays for quantifying metabolic intermediates and end products. We developed a multiplexed method for profiling CoA-dependent pathways that uses a cysteine-terminated peptide to covalently capture CoA-bound metabolites. Captured metabolites are then rapidly separated from the complex mixture by immobilization onto arrays of self-assembled monolayers and directly quantified by SAMDI mass spectrometry. We demonstrate the throughput of the assay by characterizing the cell-free synthesis of HMG-CoA, a key intermediate in the biosynthesis of isoprenoids, collecting over 10,000 individual spectra to map more than 800 unique reaction conditions. We anticipate that our rapid and robust analytical method will accelerate efforts to engineer metabolic pathways.

Sequential Photoactivation of Self-Assembled Monolayers to Direct Cell Adhesion and Migration.

Dynamic substrates for cell culture control the spatial and temporal presentation of extracellular matrix ligands that interact with adherent cells. This paper reports a photoactive surface chemistry that can repeatedly activate regions of the substrate for cell adhesion, spreading, and migration. The approach uses self-assembled monolayers presenting the integrin ligand RGD that is caged with a nitrophenyl-based photoprotecting group. The group is also modified with a maltoheptaose oligosaccharide to prevent nonspecific protein adsorption and cell attachment. The peptide is uncaged when irradiated with a laser source at 405 nm on a microscope to reveal micron-size regions for single cell attachment. This method is applied to studies of gap junction-mediated communication between two neighboring cells and requires the patterning of an initial receiver cell population and then the patterning of a second sender population to give a culture wherein each pair of cells are separated by 30 µm. Finally, activation of the region between the cells permits cell-cell contact and gap junction assembly between the sender and receiver cells. This example demonstrates the broad relevance of this method to studying complex phenotypes in cell culture.

Exploration of the nanomedicine-design space with high-throughput screening and machine learning.

Only a tiny fraction of the nanomedicine-design space has been explored, owing to the structural complexity of nanomedicines and the lack of relevant high-throughput synthesis and analysis methods. Here, we report a methodology for determining structure-activity relationships and design rules for spherical nucleic acids (SNAs) functioning as cancer-vaccine candidates. First, we identified ~1,000 candidate SNAs on the basis of reasonable ranges for 11 design parameters that can be systematically and independently varied to optimize SNA performance. Second, we developed a high-throughput method for making SNAs at the picomolar scale in a 384-well format, and used a mass spectrometry assay to rapidly measure SNA immune activation. Third, we used machine learning to quantitatively model SNA immune activation and identify the minimum number of SNAs needed to capture optimum structure-activity relationships for a given SNA library. Our methodology is general, can reduce the number of nanoparticles that need to be tested by an order of magnitude, and could serve as a screening tool for the development of nanoparticle therapeutics.

Using Peptide Arrays To Discover the Sequence-Specific Acetylation of the Histidine-Tyrosine Dyad.

Reactions that can selectively modify amino acid sequences within peptides and proteins are important for preparing protein reagents, immobilizing proteins, and making antibody-drug conjugates. The development of new reactions often begins with known chemistries and optimizes yields using a small set of peptide reactants. This article describes the use of peptide arrays and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to discover and characterize unanticipated sequence-selective reactions of peptides. This work reports the selective acetylation of HY (histidine-tyrosine) and YH (tyrosine-histidine) dyads when treated with acetic anhydride in aqueous conditions. More broadly, this example illustrates the benefits of using peptide arrays and a label-free analysis method to discover peptide-modifying reactions and gain mechanistic insight into their sequence specificity.

Photoactivatable Reaction for Covalent Nanoscale Patterning of Multiple Proteins.

This article describes a photochemical approach for independently patterning multiple proteins to an inert substrate, particularly for studies of cell adhesion. A photoactivatable chloropyrimidine ligand was employed for covalent immobilization of SnapTag fusion proteins on self-assembled monolayers of alkanethiolates on gold. A two-step procedure was used: first, patterned UV illumination of the surface activated protein capture ligands, and second, incubation with a SnapTag fusion protein bound to the surface in illuminated regions. Two different fluorescent proteins were patterned in registry with features of 400 nm in size over a 1 mm2 area. An example is given wherein an anti-carcinoembryonic antigen (anti-CEA) scFv antibody was patterned to direct the selective attachment of a human cancer cell line that express the CEA antigen. This method enables the preparation of surfaces with control over the density and activity of independently patterned proteins.

Combining SAMDI Mass Spectrometry and Peptide Arrays to Profile Phosphatase Activities.

Phosphatases, the enzymes responsible for dephosphorylating proteins, play critical roles in many cellular processes. While their importance is widely recognized, phosphatase activity and regulation remain poorly understood. Currently, there are few assays available that are capable of directly measuring phosphatase activity and specificity. We have previously introduced SAMDI (self-assembled monolayers on gold for matrix-assisted laser desorption/ionization) mass spectrometry as a technique to profile the substrate specificities of enzymes. SAMDI mass spectrometry assays are well suited to examine phosphatase activities and offer many advantages over current methods. This technique uses monolayers that terminate with a peptide or molecular enzyme substrate and allows for enzyme reactions to be performed on a surface that can easily be rinsed and analyzed by mass spectrometry without the need for analyte labeling. In this chapter, we describe the process of combining SAMDI mass spectrometry with peptide arrays to study the substrate specificities of two protein tyrosine phosphatases.