Krishnan K. Palaniappan

Thiacycloalkynes for Copper-Free Click Chemistry

Bioorthogonal chemistry enables the interrogation of biomolecules and physiological processes that are inaccessible by using conventional research tools.1 A common experimental protocol starts by labeling a target biomolecule in cells or live organisms with a bioorthogonal functional group. Then, a probe molecule bearing complementary functionality is added to the system and the ensuing bioorthogonal chemical reaction delivers the probe specifically to the targets of interest. For many applications, rapid reaction kinetics are essential. This is particularly true for labeling experiments in live animals, in which reagent concentrations are limited (i.e., nm to low μm), or in which the process that is probed occurs on a fast time scale. Consequently, methodologists interested in the development of bioorthogonal reactions are increasingly focused on kinetic optimization.2

A Xenon-Based Molecular Sensor Assembled on an MS2 Viral Capsid Scaffold

In MRI, anatomical structures are most often differentiated by variations in their bulk magnetic properties. Alternatively, exogenous contrast agents can be attached to chemical moieties that confer affinity to molecular targets; the distribution of such contrast agents can be imaged by magnetic resonance. Xenon-based molecular sensors are molecular imaging agents that rely on the reversible exchange of hyperpolarized xenon between the bulk and a specifically targeted host-guest complex. We have incorporated approximately 125 xenon sensor molecules in the interior of an MS2 viral capsid, conferring multivalency and other properties of the viral capsid to the sensor molecule. The resulting signal amplification facilitates the detection of sensor at 0.7 pM, the lowest to date for any molecular imaging agent used in magnetic resonance. This amplification promises the detection of chemical targets at much lower concentrations than would be possible without the capsid scaffold.

Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis

Protein glycosylation is a heterogeneous post-translational modification (PTM) that plays an essential role in biological regulation. However, the diversity found in glycoproteins has undermined efforts to describe the intact glycoproteome via mass spectrometry (MS). We present IsoTaG, a mass-independent chemical glycoproteomics platform for characterization of intact, metabolically labeled glycopeptides at the whole-proteome scale. In IsoTaG, metabolic labeling of the glycoproteome is combined with (i) chemical enrichment and isotopic recoding of glycopeptides to select peptides for targeted glycoproteomics using directed MS and (ii) mass-independent assignment of intact glycopeptides. We structurally assigned 32 N-glycopeptides and over 500 intact and fully elaborated O-glycopeptides from 250 proteins across three human cancer cell lines and also discovered unexpected peptide sequence polymorphisms (pSPs). The IsoTaG platform is broadly applicable to the discovery of PTM sites that are amenable to chemical labeling, as well as previously unknown protein isoforms including pSPs.

Molecular Imaging of Cancer Cells Using a Bacteriophage‐Based 129Xe NMR Biosensor

The accurate detection and localization of clinically relevant biomarkers in vivo is a great challenge for molecular imaging, requiring high sensitivity and molecular specificity. This is particularly true for screening applications, where the ability to image disease progression non-invasively could improve patient outcome. Magnetic resonance imaging (MRI) is a ubiquitous, non-invasive imaging technique with sub-millimeter spatial resolution, but its use in molecular imaging has been limited by its poor sensitivity when imaging molecules other than water. This has led to the development of contrast agents and MRI methods that improve sensitivity by modulating the local magnetic environment of protons in water, including gadolinium chelators, iron-oxide particles, and chemical exchange saturation transfer (CEST). More recently, approaches that use hyperpolarized C, He, and Xe nuclei have been developed and used in clinical studies. In the experiments below, we used xenon (Xe) as a sensor medium. Xe is an attractive option for MRI-based molecular imaging because it is chemically inert, has low toxicity, is soluble in water and tissue, and can be hyperpolarized (hp) to increase its signal more than 10 000-fold. Thus, even low concentrations of dissolved Xe give an NMR signal comparable to that of water, and there is no Xe background in vivo. These favorable properties of Xe MRI have already been demonstrated in humans after inhalation of hp Xe gas. Molecular imaging agents that leverage these characteristics, generally called Xe biosensors, have been developed. They consist of a Xe-binding host molecule, commonly cryptophane-A (CryA), attached to targeting groups for localization. Xenon bound by CryA (Xe@CryA) has a distinct chemical shift apart from that of aqueous Xe (Xe@water). Its rapid and reversible encapsulation is the basis of an indirect detection scheme in which the small Xe@CryA spin pool is saturated by frequency-selective radiofrequency (RF) pulses and transferred by exchange to the larger Xe@water spin pool, an example of amplification by CEST. This results in a decrease in the signal relative to a control experiment (Figure 1a). Combined with the signal enhancement of hyperpolarization, Xe biosensors can achieve the detection thresholds necessary for molecular imaging. To improve the sensitivity further we used multivalent systems in which many CryA hosts are assembled onto a single carrier molecule, a concept initially applied with paramagnetic relaxation and CEST agents. We have demonstrated this strategy for Xe biosensors with branched dendrimers and viral capsids, producing constructs that were detectable by hyperCEST at sub-picomolar concentrations. In previous studies of Xe biosensors biological binding events were measured in solution. This was first achieved with biotin-functionalized biosensors binding streptavidin beads, 14a,18] and subsequently with the detection of DNA hybridization, enzymatic cleavage by matrix metalloproteinase-7, ligand binding to human carbonic anhydrase and an a2bb3 integrin, [22] and peptide complex formation with a major histocompatibility complex protein. Once the cellular compatibility of Xe biosensors was established, Xe NMR spectroscopy was performed with cells after targeting them with micromolar concentrations of a transferrin-functionalized biosensor. While that study detected biosensor binding by measuring the Xe@CryA chemical shift, non-specific binding was also observed. Here, we report a multivalent Xe biosensor that uses single-chain antibodies to target cell surface biomarkers. We further specifically demonstrate its ability to specifically recognize these biomarkers in living cells and at concentrations required for molecular imaging. To accomplish this, we used fd filamentous bacteriophage that display single-chain antibody variable fragments (scFvs) on their minor coat proteins (p3, Figure 1 b). The rod-like body of the fd phage, which has 4200 identical copies of the major coat protein (p8), can be modified with proteins or synthetic molecules to create new materials. Additionally, through the use of phage display techniques, filamentous phage that display proteins as [*] Dr. K. K. Palaniappan, R. M. Ramirez, Dr. V. S. Bajaj, Prof. Dr. D. E. Wemmer, Prof. Dr. A. Pines, Prof. Dr. M. B. Francis Department of Chemistry, University of California Berkeley, CA 94720-1460 (USA) E-mail: mbfrancis@berkeley.edu R. M. Ramirez, Dr. V. S. Bajaj, Prof. Dr. A. Pines, Prof. Dr. M. B. Francis Materials Sciences Division Lawrence Berkeley National Laboratory Berkeley, CA 94720-1460 (USA)

A Chemical Method for Labeling Lysine Methyltransferase Substrates

Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin‐dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome‐wide identification of novel PKMT substrates. Our strategy makes use of an alkyne‐bearing S‐adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide‐functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof‐of‐concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

HyperCEST detection of a 129Xe-based contrast agent composed of cryptophane-A molecular cages on a bacteriophage scaffold.

A hyperpolarized 129Xe contrast agent composed of many cryptophane‐A molecular cages assembled on an M13 bacteriophage has been demonstrated. Saturation of xenon bound in the large number of cryptophane cages is transferred to the pool of aqueous‐solvated xenon via chemical exchange, resulting in efficient generation of hyperCEST contrast. No significant loss of contrast per cryptophane cage was observed for the multivalent phage when compared with unscaffolded cryptophane. Detection of this phage‐based hyperCEST agent is reported at concentrations as low as 230 fM, representing the current lower limit for NMR/MRI‐based contrast agents. Magn Reson Med, 2013.

MRI thermometry based on encapsulated hyperpolarized xenon.

A new approach to MRI thermometry using encapsulated hyperpolarized xenon is demonstrated. The method is based on the temperature dependent chemical shift of hyperpolarized xenon in a cryptophane-A cage. This shift is linear with a slope of 0.29 ppm °C(-1) which is perceptibly higher than the shift of the proton resonance frequency of water (ca. 0.01 ppm °C(-1)) that is currently used for MRI thermometry. Using spectroscopic imaging techniques, we collected temperature maps of a phantom sample that could discriminate by direct NMR detection between temperature differences of 0.1 °C at a sensor concentration of 150 μM. Alternatively, the xenon-in-cage chemical shift was determined by indirect detection using saturation transfer techniques (Hyper-CEST) that allow detection of nanomolar agent concentrations. Thermometry based on hyperpolarized xenon sensors improves the accuracy of currently available MRI thermometry methods, potentially giving rise to biomedical applications of biosensors functionalized for binding to specific target molecules.

Mapping Yeast N-Glycosites with Isotopically Recoded Glycans

Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.

Isotopic Signature Transfer and Mass Pattern Prediction (IsoStamp): An Enabling Technique for Chemically-Directed Proteomics

Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide’s mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide’s sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.