Program

Sensors for biological measurements could change the way that we currently measure analytes in the body, but the design requirements for implementing these tools in vivo are challenging. Among the obstacles that sensors face include fouling, specificity, and biocompatibility/foreign body response. A variety of sensors, including electrochemical and optical, have progressed significantly to address these challenges. This symposium will highlight sensing technologies that have made major advances in long-term sensing in vivo.

Sensing Science in Brain Chemistry

Development of new sensing strategies and methodologies to directly, selectively, and sensitively record chemical signals of neurons during brain functions has drawn increasing attention because information on the dynamics of chemical signals is very essential to understanding the chemical essence involved in brain functions, for example, neurotransmission and diagnosis and therapy of brain diseases. However, the chemical and physiological complexity of the central nervous system (CNS) unfortunately make this pursuit very challenging to the conventional sensing/analytical protocols. Aiming at this challenge, we have been working on sensing science in live brain ranging from mechanistic development (mainly with rationally modulating electrode/brain interface) to in vivo understanding brain chemistry. This topic will focus on our recent attempts on sensing science in live brain based on rational design and regulation of electrode/brain interface and its application for in vivo understanding brain chemistry.

3D Printed electrodes for neurotransmitter measurements

Would you like to build a better carbon microelectrode from the ground up? Microelectrodes are the standard method for measuring neurotransmitters in vivo, but their design has been based on carbon fibers, which limits the geometries possible.  Here, we introduce a novel, implantable and freestanding microsensor fabrication method using two-photon nanolithography followed by pyrolysis.  This 3D printing method allows the fabrication of free-standing carbon microelectrodes with customizable geometry and electroactive carbon surface, which is suitable for neurotransmitter detection. Pyrolysis carbonizes and shrinks the photoresist, so this method is useful for making nanoelectrodes and allows batch fabrication of sensors. We have demonstrated the use of these electrodes in vivo and in Drosophila for measurements of neurotransmitters.

Imaging Brain Neuromodulation with Near-Infrared Fluorescent
NanosensorsNeurons communicate through chemical neurotransmitter signals that either terminate at the postsynaptic process (“wired transmission”) or diffuse beyond the synaptic cleft to modulate the activity of larger neuronal networks (“volume transmission”). Molecules such as dopamine, serotonin, and neuropeptides such as oxytocin belong to the latter class of neurotransmitters, and have been the pharmacological targets of antidepressants and antipsychotics for decades. Owing to the central role of neuromodulators such as dopamine over a range of behaviors and psychiatric disorders, real-time imaging of the signal’s spatial propagation would constitute a valuable advance in neurochemical imaging. To this end, we present a library of nanoscale near-infrared fluorescent nanosensors for dopamine, serotonin, and oxytocin, where the nanosensors are developed from polymers pinned to the surface of single wall carbon nanotubes (SWNT) in which the surface-adsorbed polymer is the recognition moiety and the carbon nanotube the fluorescence transduction element. Excitonic transitions in functionalized SWNT yield up to ΔF/F = 4500% near-infrared fluorescence emission in the presence of dopamine (Beyene et al. Nano Letters 2018), ΔF/F = 200% for serotonin (Jeong et al. Science Advances 2019), and ΔF/F = 120% for oxytocin (unpublished). We next demonstrate imaging of evoked dopamine release in acute striatal slices, and show altered dopamine reuptake kinetics when brain tissue is exposed to dopamine receptor agonist and antagonist drugs (Beyene et al. Science Advances 2019). We characterize our findings in the context of their utility for high spatial and temporal neuromodulator imaging in the brain, describe nanosensor exciton behavior from a molecular dynamics (MD) perspective, and validate nanosensor for use to elucidate dopaminergic signaling variability with disease or pharmacological perturbations at a synaptic scale.

Engineering the Nanoparticle Corona and Optical Techniques for In-Vivo Sensing at Biological Interfaces

Nanosensors, particularly those based on nanoparticle transducers, have demonstrated the ability to detect the binding of molecules at the single analyte level.  Our lab at MIT has been interested in how the nanoparticle corona – the region of adsorbed molecules surrounding the particle surface – can be engineered for molecular recognition.  We have recently introduced a method we call CoPhMoRe or Corona Phase Molecular Recognition¹ for discovering synthetic, heteropolymer corona phases that form molecular recognition sites at the nanoparticle interface, selected from a heteropolymer library. We show that certain synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for specific molecules.  We have a growing list of biomolecules that we can detect using this approach including riboflavin, L-thyroxine, dopamine, nitric oxide, sugar alcohols, estradiol, as well as proteins such as fibrinogen.  The results have significant potential in light of the fact that nanoparticles such as single walled carbon nanotubes can be interfaced to biological systems at the sub-cellular level, with unprecedented sensitivity.  Several recent demonstrates indicate that spatial and temporal information on cellular chemical signaling can be obtained using arrays of such sensors.  To enable the use of these types of sensors, we have developed techniques that allow optical excitation to propagate through the in-vivo environment, avoiding unfavorable tissue scattering and intrinsic autofluorescence. We develop a wavelength-induced frequency filtering (WIFF) whereby the fluorescence excitation wavelength is modulated across the absorption cross-section of the fluorescent bisensor, allowing the emission signal to be separated from the autofluorescent background, increasing the desired signal relative to noise, and internally referencing it to protect against artifacts. Using highly scattering tissue phantoms, an SKH1-E mouse model, and other complex tissue types, we show that WIFF significantly improves the in vivo signal to noise ratio (SNR) of fluorescent sensors up to 52 fold for commonly employed chromophores in the visible spectrum. WIFF extends measurements to extremely deep implants up to 5.5±0.1 cm depth in chicken breast tissue when using probes excited at 730 nm and emitting between 1150 and 1300 nm, even allowing the monitoring of riboflavin diffusion in thick tissue. As an application, we demonstrate that WIFF enables the detection of the chemotherapeutic activity of temozolomide trans-cranially 2.4±0.1 cm through the porcine brain without the use of fiber optic or cranial window insertion. These results based on WIFF open up new avenues of biomedical research by extending a large number of fluorescent sensors to previously inaccessible in vivo environments.

The SARS-CoV-2 pandemic demonstrates the need for measurement tools for rapid and accurate diagnostics, remote measurements and fundamental studies on virus structure and function. This symposium highlights recent advances in sensing, microfluidics, sequencing, mass spectrometry and optical spectroscopy applied to virus diagnostics and characterization with an emphasis on SARS-CoV-2.

Solid-State Nanopore Platform Integrated with Machine-Learning for Digital Diagnosis of Virus Infection

The variability of bioparticles remains a key barrier to realizing the competent potential of nanoscale detection into a digital diagnosis of an extraneous object that causes an infectious disease. We developed label-free virus identification based on machine-learning classification. Single virus particles were detected using nanopores, and resistive-pulse waveforms were analyzed multilaterally using artificial intelligence. In the discrimination, over 99% accuracy for five different virus species, including influenza and corona virus species, was demonstrated. This advance is accessed through the classification of virus-derived ionic current signal patterns reflecting their intrinsic physical properties in a high-dimensional feature space. Moreover, consideration of viral similarity based on the accuracies indicates the contributing factors in the recognitions.  We also developed a portable robust ionic current sensor using a bridge circuit that offers a high signal-to-noise (S/N) ratio by suppressing background current as a useful tool for detecting sub- to several-micron scale particles such as virus and bacteria.  Because the portable robust ionic current sensor can tolerate increased noise in current sensing, a simple, lightweight electromagnetic shield can be used and measurements under large electromagnetic noise conditions can be made.  This sensor combined with machine-learning system enables us to identify several virus species and several bacteria with over 90 % accuracy.

Mass spectrometry: From plasma proteins to mitochondrial membranes

Beginning with the preservation of the first soluble complexes from plasma in the gas phase of a mass spectrometer, I will describe our early experiments that capitalise on the heterogeneity of subunit composition during assembly and exchange reactions. To assess the overall topology of these complexes we adapted ion mobility and soft-landing methodologies to show how ring-shaped complexes could survive the phase transition. The next logical progression from soluble complexes was to membrane protein assemblies but this was not straightforward. We encountered many pitfalls along the way, largely due to the use of detergent micelles to protect and stabilise these complexes. Further obstacles presented when we attempted to distinguish lipids that co-purify from those that are important for function. Developing new experimental protocols, we have subsequently defined lipids that change protein conformation, mediate oligomeric states, and facilitate downstream coupling of G protein-coupled receptors. Recently, using a new method—ejecting protein complexes directly from native membranes into mass spectrometers—we provided insights into associations within membranes and mitochondria.  I will trace the history of these developments in my presentation and also look towards future innovations and discoveries.

Molecular Imprints for Selective Virus Capture: Scheme or Scam?

Molecularly imprinted polymers (MIPs) have come a long way, and have proven their utility for generating synthetic receptor materials selectively binding small molecules. How about large molecules such as proteins? Or entire biological specimen such as viruses? Potential and challenges of MIPs for selectively binding these species will be discussed to answer the provocative question asked in the title.

Ultrasensitive Immunoassays for Understanding SARS-CoV-2 Infections

Using single-molecule arrays, we have developed digital ELISAs with 2-3 logs better analytical sensitivity than conventional immunoassays.  We have used these assays to measure viral proteins, host antibodies and host inflammatory responses to SARS-CoV-2 infections.  The ultrasensitivity allows us to measure proteins earlier during infection.  Furthermore, the ability to measure extremely low concentrations enables us to observe aspects of disease pathogenesis that provide mechanistic insight.  In this talk, I will describe some of our findings that elucidate the development of immune responses and disease severity, as well as features of the host response to mRNA vaccines.

Advancing proteomic technology is improving the ability to study the proteome to learn new biology and to aid in the diagnosis of disease. This symposium will probe new strategies to identify and quantify posttranslational modifications to determine their biological roles. The speakers will encompass bottom- up strategies as well as top down to identify PTMs as well as proteoforms.

Most studies have established macrophages as an indispensable key player in both innate immune responses and adaptive immunity. Thus, they are ideal therapeutic targets for diseases such as insulin resistance, cancer, type 2 diabetes, atherosclerosis, and periodontitis. Macrophages are not homogenous, and they are generally categorized into two broad but distinct subsets as either classically activated (M1, pro-inflammatory) or alternatively activated (M2, anti-inflammatory). Recent studies have demonstrated that macrophages produce extracellular traps (ETs). ETs are an immune response by which a cell undergoes “ETosis” to release net-like material, with strands composed of cellular DNA that is studded with histones and cellular proteins. Peptidylarginine deiminase 4 (PAD4) is critically involved in chromatin decondensation during the release of ETs. The aim of our study was to investigate the role of PAD enzymes and its downstream effects, such as the protein citrullination in polarization of macrophages to M1 and M2. We used human monocytic cell line THP-1 differentiated by PMA to macrophages. The cells were further differentiated to proinflammatory M1 and anti-inflammatory M2 macrophages by 10 ng/ml LPS (S. Minnesota) and 20 ng/ml IL-4, respectively, and treated with 100 nM PAD inhibitor – BB-Cl-amidine for 48 h. We measured mRNA expression of different PAD isoforms and M1 and M2 markers upon treatment with BB-Cl-amidine. To identify differentially changed proteins and protein citrullination sites we applied a data independent mass spectrometry method (DIA) and hyper-citrullinated spectral library approach. We determined that PAD inhibitor decreased expression of proinflammatory M1 markers and increased expression of anti-inflammatory M2 markers in THP-1 macrophages. PAD2, the most abundant PAD in macrophages, was downregulated in M2 macrophages. Citrullination seems to play a major role in proinflammatory M1 macrophages with several citrullinated proteins involve in platelet activation, signaling and aggregation. The study showed that PADs and protein citrullination could play a critical role in the differentiation of macrophage and inflammatory response. Furthermore, identified citrullinated proteins, could be the new autoantigen that influence anti-citrullinated protein antibodies and can be used as a new marker for disease detection and progression.

Histone Proteoform Mechanisms of Epigenetic Regulation and Dysregulation in Aging and Disease

The physiological state of the human genome is chromatin, a nucleo-protein complex consisting of approximately 50% protein. Histone proteins package the genome into nucleosomes, help regulate gene expression, and help coordinate genome maintenance. These functions are achieved by the use of multiple sequence variants of histone proteins carrying complex single molecule combinations of post-translational modifications (PTMs) or more succinctly ‘histone proteoforms’. This proteoform-based system is most effectively addressed by quantitative top down proteomics. We have developed high throughput quantitative top down proteomics methods to rigorously quantitate hundreds of histone proteoforms as low as 10 ppm abundance starting from limited cells or tissues. Initial applications in cell culture systems have revealed novel proteoform-based mechanisms of genome regulation and novel therapeutic strategies in cancer. We now extend this work into in vivo neuroepigenetics. We present the first spatially resolved quantitative atlas of histone proteoforms resolved between 11 different regions of the C57BL/6 mouse and a series of experiments probing how these proteoforms change with age. Histone PTMs and proteoforms vary significantly between brain regions and temporally during development and aging. These insights are being applied to multiple in vivo models of disease of aging and Alzheimer’s disease to connect this histone proteoform biology to upstream kinase and transcription factor signaling and downstream to lysosomal biogenesis and cellular health.

Applications of Data-Independent Acquisitions to Quantify Post-translational Modifications and to Monitor their Dynamic Signaling during Aging and Disease.

Recent work is presented using data-independent acquisitions (DIA) to identify and quantify PTM containing peptides in a high-throughput format. Several projects will be presented assessing protein acylation, such as acetylation and succinylation in liver and brown adipose fat tissues, and other PTMs. Methodologically, we will present simultaneous enrichment of multiple PTMs using antibody enrichment, and apply DIA-MS workflows to assess PTM crosstalk and to obtain comprehensive PTM coverage. PTM site localization is determined using various software tools, such as Skyline and Spectronaut. Acquiring PTM samples in DIA mode enables capabilities to identify multiple different site localization isomers from peptides with the same peptide sequence and precursor ion m/z. DIA acquisitions for PTM profiling and quantification provide unique opportunities to overcome challenges that are associated with protein PTM workflows. To decipher dynamic PTM changes and PTM signaling, we will discuss the relevance and protective role of succinylation during acute kidney injury, the role of succinylation as part of brown adipose tissue function, and the extremely dynamic and massive remodeling of the acylome and succinylome in liver upon dietary supplementation.

New Technologies for Exploiting the Human Glycoproteome for Cardiovascular Physiology and Personalized Medicine 

Cell surface glycoproteins and glycans play critical roles in a range of biological functions and disease processes and may be exploited as biomarkers for precision medicine. Despite their biological relevance and utility, glycoproteins and glycans are often understudied largely due to technical challenges. This presentation will describe our recently developed analytical and bioinformatic solutions that enable rapid identification and quantification of cell surface glycoproteins and glycans from small sample sizes. The application of these new methodologies to address outstanding questions in cardiac biology and disease, with an emphasis on precision medicine, will be described.

Latest Applications of 4D-Proteomics using the timsTOF Pro and recent developments in ultra-high sensitivity and single cell proteomics

This session will discuss the PASEF, dia-PASEF and prm-PASEF methods and explain how these methods allow researchers to perform proteomics faster, with better depth of coverage, better sensitivity, and with ore confidence in their identification and quantitation.

After a summary of these methods, this session will cover some of the latest applications of the timsTOF Pro and 4D-proteomics technology to biomarker discovery and validation, immunopeptidomcs and single cell proteomics.

Topics

– Features and performance of 4D-proteomics on the timsTOF Pro

– PASEF, dia-PASEF, prm-PASEF

– High throughput biomarker discovery and validation

– Ultra-high sensitivity for immunopeptidomics and single cell proteomics

BRUKER FM: Far and Mid IR Spectral Range Spectroscopy in One Step

Featured Speaker
Gary Kruppa, Ph.D., Vice President of Proteomics, Bruker Daltonics Inc., has over 30 years of experience in the field of mass spectrometry (MS), having served as a Vice President at Bruker Daltonics for over 20 years.

Kruppa received his Ph.D. in chemical physics from the California Institute of Technology, and his BS from the University of Delaware. Kruppa oversees market and applications development management for Bruker’s innovative solutions for research in proteomics.

From the development of ESI and MALDI ionization methods over 30 years ago, mass spectrometry (MS) has been at the forefront of contributing to advancements and uncovering new knowledge in all areas of science. This symposium will highlight current and future cutting-edge MS technologies that offer advances in ionization methods and sampling, analyte separation (both liquid-phase and gas-phase), and mass measurement platforms, to make MS measurements of biological molecules more sensitive and selective than in the past, potentially impacting biology and human health research.

Chemical Measurements In Vivo with the MasSpec Pen Technology

Implementation of new technologies that provide precise molecular diagnosis of tissues are highly desirable to guide treatment strategies and improve cancer patient care. Molecular technologies offer the exciting opportunity to incorporate cancer-specific markers into clinical decision making for improved cancer detection and diagnosis. In particular, ambient ionization mass spectrometry (MS) techniques provide the specificity and sensitivity necessary to perform in situ analysis of tissues for near real time assessment of their molecular signatures. In my presentation, I will describe my lab’s research developing and translating the MasSpec Pen technology for direct molecular analysis of in vivo and freshly excised tissues. The MasSpec Pen enables gentle extraction and sensitive detection of various molecular species including small metabolites and lipids using a droplet of water without causing tissue damage. When implemented by surgeons in the operating room, rapid detection of rich mass spectral profiles from in vivo and ex vivo analyses performed on tissues during over 100 cancer surgeries was achieved. Collectively, our current results provide evidence that the MasSpec Pen can be successfully incorporated into the operating room, allowing direct detection of molecular profiles from tissues with a seconds-long turnaround time that could be transformative to inform surgical and clinical decisions.

New Frontiers of Ultraviolet Photodissociation Mass Spectrometry for Characterization of Biological Molecules

Advances in mass spectrometry instrumentation and experimental design have led to significant inroads in the characterization of biological molecules, from lipids to protein complexes. Ultraviolet photodissociation (UVPD) has emerged as a high energy ion activation method that results in extensive fragmentation and helps uncover structural details of molecules, like mutations, modifications, and locations of sites of unsaturation. UVPD also offers a versatile MS/MS technology for characterization of intact proteins, including determining disulfide bonds and ligand binding sites. There has been growing interest in employing MS/MS strategies to examine native protein structures by disassembling multimeric complexes. The fast, high energy deposition of UV photoactivation allows retention of non-covalently bound ligands and generates sequence ions that identify the constituent proteins. The performance of UVPD and other MS/MS methods is further extended by integrating these methods with gas-phase separation methods, including ion mobility using modular drift tubes, as well as gas-phase reactions such as ion-ion proton transfer reactions that manipulate charge states of ions.

High dimensional molecular phenomics in systems, synthetic, and chemical biology: Peering into the unknown

One of the predominant challenges in systems-wide analyses and molecular phenomics is the broad-scale characterization of the molecular inventory in cells, tissues, and biological fluids. Advances in computational systems biology rely heavily on the experimental capacity to make omics measurements, i.e. integrated metabolomics, proteomics, lipidomics, glycomics, etc., accompanied with fast minimal sample preparation, fast measurements, high concentration dynamic range, low limits of detection, and high selectivity. This confluence of figures-of-merit place demanding challenges on analytical platforms for such analyses. Ion mobility-mass spectrometry (IM-MS) provides rapid (ms) gas-phase electrophoretic separations on the basis of molecular structure and is well suited for integration with rapid (us) mass spectrometry detection techniques. This presentation will describe recent advances in IM-MS integrated omics measurement strategies in the analyses of complex biological samples of interest in systems, synthetic, and chemical biology in clinical chemistry applications. New advances in bioinformatics and biostatistics will also be described to approach biological queries from an unbiased and untargeted perspective and to quickly mine the massive datasets gathered to provide targeted and actionable information.

New horizons in Top-Down and Single-Particle Mass Spectrometry

Native mass spectrometry enables both the mass analysis of intact assemblies into the megadalton range as well as the detailed structural analysis of large intact proteins decorated by a plethora of post-translational modifications. In this presentation, I will describe remaining challenges in the field, and highlight some of the advances made to tackle those, including extending the mass range, the sensitivity, the mass resolving power and the specificity of native mass spectrometry.

In the first part I introduce Orbitrap-based charge detection single molecule mass spectrometry. A limitation in native mass spectrometry stems from the fact that the charge state, and thus mass, can only be measured when multiple charge states can be resolved/assigned. This hampers the analysis of heterogeneous assemblies. A potential solution is to measure one particle at a time, thereby avoiding the convolution of ion signals. The Orbitrap mass analyser is sufficiently sensitive to detect single ions. We demonstrate that the intensity of a single ion in an Orbitrap can be used to infer its charge state, and thus mass. This enables the sensitive analysis of heterogeneous assemblies like immunoglobulins, ribosomes, proteinaceous nanocontainers and genome packed adeno-associated viruses. The latter is important for quality control of therapeutically used gene-delivery vectors.

In the second part I describe the analysis of serum derived intact glycoproteins by high-resolution native mass spectrometry. Our serum contains many abundant glycoproteins, many of which have been implicated as biomarkers. However, most serum glycoproteins are characterized by a plethora of proteoforms with putatively different functions. I will show that comprehensive proteoform analysis has become feasible with recent advances in mass spectrometry. Unlike most clinical assays, these MS-based approaches enable us to monitor not only protein abundance, but also proteoform abundance. This allows us to address important questions such as; Can we quantitatively measure serum glycoprotein proteome profiles of single donors? Can we monitor how serum glycoprotein proteome profiles respond to changes in a donors’ physiological state? Can the latter aid in improving personalized diagnostics and therapies? And finally the fundamental question; How unique is our proteome?

The Future of Data Management: How FAIR Principles and Data Harmonization Will Transform the Lab

The ways we capture, store, and retrieve analytical data are becoming increasingly complex—proprietary data silos and the lack of bidirectional communication between instruments are becoming major barriers to efficiency, automation, and remote collaboration.

In this presentation, we will review the importance of FAIR data principles (Findable, Accessible, Interoperable, and Reusable) and review how applying data harmonization through open standards such as AnIML (Analytical Information Markup Language) enable transformational improvements to long-term data management and data governance.

Topics Covered:

– Overview of the state of data management today and barriers to progress

– Review the business and operational implications of FAIR data principles 

– Introduction to existing open data standards and the benefits of AnIML and SiLA

Speakers:

Haydn Boehm, Head of Commercial Marketing, Connected Lab, MilliporeSigma

Haydn Boehm is the Head of Commercial Marketing for MilliporeSigma’s Connected Lab digital program and has over 20 years of experience in marketing and business development within the life scientific sector. Haydn has a Ph.D. in Organic Chemistry from the University of Nottingham, UK.

Annette Hellbach, Head of Product Management, Connected Lab, MilliporeSigma

Annette Hellbach is the Head of Product Management at MilliporeSigma’s Connected Lab. Annette has a Ph.D. in Molecular Genetics from the Max Planck Institute of Biochemistry/Ludwig-Maximilians-University Munich. She drove 3rd party instrument integration and business development in Thermo Fisher’s lab informatics business and worked with Pharma customers on data and IT strategy projects before joining MilliporeSigma’s Connected Lab.