Areas of Interest
The Coon Research Group has the overarching goal of catalyzing evolution in the rapidly growing field of proteomics and using these technologies to address fundamental problems in developmental biology. With emphasis on ion chemistry and instrumentation, we seek to develop and apply new enabling mass spectrometry-based (MS) proteomic technologies. These cutting-edge tools allow us to examine, with unprecedented chemical detail and sensitivity, the molecular events that commit human embryonic stem cells (hES cells) to exit the pluripotent state. Here we focus on both intracellular signaling and the epigenetic regulation of pluripotency. For the former we ask which branches of the FGF signaling pathway are active in hES cells and which proteins/networks are phosphorylated upon differentiation. Epigenetics is believed to play a critical role in the establishment and maintenance of pluripotency; thus, we have also aimed our new technologies at interpreting the epigenetic codes and monitoring message changes during hES cell differentiation.
Informatics
We develop algorithms and software to deal with all aspects of post-acquisition data analysis. Much of our workflow is based around the Open Mass Spectrometry Search Algorithm (OMSSA), which is open-source, fast, scalable, flexible, easy to use, and produces excellent results for a variety of common proteomics tasks. These tasks include data reduction from proprietary instrument vendor file formats to text-based input file formats for database searching, peptide false discovery rate determination, peptide quantification, modification (particularly phosphorylation) localization, protein parsimony and false discovery rate determination, protein quantitation, and modification quantitation.
Instrumentation
The goal of the lab is to advance electron transfer dissociation (ETD)-equipped mass spectrometer instrumentation. This goal encompasses: (1) upgrading and optimizing mass spectrometer equipment already capable of performing ETD, and (2) adapting additional mass spectrometer equipment to be compatible with ETD. We have achieved two significant successes – one for each sub-category. During late spring 2006, the lab converted non-dissociated electron transfer (ET) products into c- and z-type ions (i.e. the typical ETD product ions). Graeme McAlister adapted the instrument (an LTQ equipped with a prototype Thermo Electron ETD package) for gentle CAD of the electron transfer product. At low activation energy, the ET product ions could be converted in c- and z-type ions without generating typical CAD ions, which would have cluttered the spectra. By adding in this additional activation step, Danielle Swaney was able to generate additional fragment ions and produce far more informative spectra.
In fall 2006, Graeme McAlister adapted an LTQ-Orbitrap hybrid mass spectrometer to allow for ETD. As evinced by the resulting spectra, mass analysis with an Orbitrap produces significantly more resolved spectra spectra with better mass accuracy, allowing for more successful fragmentation identification. In particular, highly charged fragment ions could now be identified which would have been impossible with QLT mass analysis. Due to spatial complications, a CI source could not be mounted on the rear of the instrument (i.e. how the current LTQ's have been adapted to allow for ETD). Drawing heavily from previous research done by the McLuckey group at Purdue, Graeme instead used the same atmospheric pressure inlet, on the front of the instrument, to inject both the cations and the reagent anions. To utilize the same inlet for both ions, the sources had to be pulsed.
Ion-Ion Reactions
Proteins are involved in nearly every aspect of cellular function. In fact, the characterization of proteins has become such a significant part of modern biology, it has inspired a new discipline: proteomics – the classification of the protein complement expressed by the genome of an organism. Technology development has, and continues, to drive rapid evolution in this field. We use linear quadrupole ion trap mass spectrometers to perform ion/ion reactions – reactions of small-molecule anions with peptide/protein cations in the gas phase – for protein characterization. In general, these reactions can be classified in three categories:
(1) reactions that remove charge from the peptide (proton transfer)
(2) reactions that transfer an electron to the peptide (electron transfer dissociation)
(3) reactions that result in the formation of a complex (anion attachment)
We study the first two of these reactions and use them alone or in sequence to identify and characterize proteins on a global-scale (proteomics).
Sequencing Peptides
The electron transfer reaction results in the attachment of an electron to the protonated peptide. The odd-electron peptide then undergoes very rapid (femtosec) rearrangement with subsequent dissociation of the N – C bond. The process occurs efficiently and randomly across the entire sequence of peptides and whole proteins. With a resulting collection of peptide fragment ions that differ in mass by a single amino acid, one can read the amino acid sequence of the peptide. And, by repeating this sequence in a rapid, automated fashion (3- 4 analyses/sec), we can characterize several hundred peptides as they elute from a nanoflow HPLC separation column (~ 2 hour gradient). Most proteomics strategies use enzymes to cleave the analyte proteins into small peptides – peptides that are then sequenced with mass spectrometers. As an alternative method, we are pursuing the use of our technology to directly interrogate intact proteins in the gas-phase. By performing our experiment in the context of the whole protein, we can begin to elucidate important biological events such as global patterns of modification and protein-alternative splicing events.
Projects
Proteomics of mitochondrial dysfunction in type 2 diabetes
The prevalence of obesity-linked type 2 diabetes mellitus (T2D) has reached epidemic proportions in the United States, as well as in other countries around the world. There is significant evidence that mitochondrial dysfunction in metabolic tissues plays an integral role in T2D, but little is understood about the underlying mechanisms. Through collaboration with the laboratories of David Pagliarini and Alan Attie, we are using quantitative mass spectrometry to map alterations of the mitochondrial proteome with the onset of T2D in animal models. By comparing the mitochondrial proteomes of mice which are resistant to diabetes (C57BL/6 leptin ob/ob) and mice which develop severe diabetes (BTBR leptin ob/ob), our goal is to identify cellular events contributing to mitochondrial dysfunction with the onset of T2D. Through a systems biology approach, we are integrating our data with those of previous microarray studies to determine the role of post-transcriptional gene regulation in the remodeling of the mitochondrial proteome. With the use of cutting-edge phosphoproteomic technologies, we also aim to decipher signaling pathways important for maintaining resistance to T2D.
The epigenotype of pluripotency
This research is based on the hypothesis that human ES cells are distinguished from their somatic cell counterparts by unique epigenotypes. The new technology created by our laboratory has allowed for the first high fidelity examination of human ES cell histone modification patterns. Ongoing aims include the global characterization and quantification of the full repertoire of PTM patterns within the core histone tails of human ES cells relative to human control cells. And, using potent differentiating agents, we seek to determine the changes to those histone codes upon lineage commitment.
Gas-phase coordination chemistry for rapid, robust whole protein sequencing
This project endeavors to describe gas phase chemistries that utilize small molecule anions that covalently bind to selected sites of a gas phase protein cation (e.g. selected amino acids). The bound anionic reagents will then serve as scaffolds to harbor site-specific cleavage with enzyme-like specificity. In this manner we can digest a large protein into smaller peptides that are more amenable to mass spectrometric analysis. This project has been funded by the Beckman Young Investigator Program.
Negative electron transfer dissociation
In this project, we intend to evolve negative electron transfer dissociation into a robust fragmentation method for large scale LC-MS/MS analysis. We will characterize the negative electron transfer dissociation method and the role the reagent cation plays in the fragmentation technique. This information will guide us in selecting the reagent cation that allows for the greatest NETD sensitivity (i.e. the highest precursor to product conversion efficiency). Also, we will optimize the LC-MS/MS NETD workflow by developing chromatography methods that work well with negative ionization, by devising AGC techniques that work well with the participating ions, and by adapting the database search algorithms to search for NETD specific fragment ions.
Sequential ion/ion reactions for large peptide and whole protein sequence analysis
The aim of this project is to develop a suite of ion/ion reaction chemistries, and to automate their use in a novel MS system. These ion/ion chemistries will be strung together to create original interrogation methods for large peptide and whole protein sequence analysis. These chemistries will be based around the three fundamental types of ion/ion reactions (i.e. proton transfer, electron transfer, and ion attachment), and will utilize an array of reagent molecules. This work is funded by the NIH (RO1 GM080148) and by Thermo Fisher Scientific through a sponsored research agreement.
Medicago truncatula phosphoproteomics
Legumes are an extremely important family of plants due to their ability to fix atmospheric nitrogen through symbiosis with rhizobia bacteria. The initiation and maintenance of legume/rhizobia symbiosis requires protein phosphorylation-mediated signaling in the root cells of the plant in response to chemicals secreted by the bacteria. Therefore, characterizing the sites of phosphorylation on legume proteins is important for fully understanding this process. Through collaboration with other members of the Wisconsin Medicago Group, we recently reported 3,404 sites of in vivo protein phosphorylation within roots of the model legume Medicago truncatula (Grimsrud et al., Plant Physiology 2010, 152: 19-28.). Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis. Furthermore, as this work was the first plant phosphoproteomic study to utilize the novel methodology of ETD, phosphorylation motifs were identified which were not previously observed in plants. These data were used to create an open-access on-line database for M. truncatula phosphoproteomic data. One ongoing aims is the utilization of quantitative phosphoproteomics to monitor symbiosis signal transduction.
Quantifying stress-activated changes of protein abundance in S. cerevisiae
The ability to sense, respond to, and combat stress factors is a fundamental survival ability of all organisms. Yeast is a model organism that allows us to study the effects of standard environmental stresses, elucidating acquired resistance and epigenetic memory resistance. Under certain stressors, yeast cells have the ability to retain “memory” of stress resistance, even upon removal from stress factors. The coupling of isobaric TMT tags and a high-resolution LTQ Orbitrap Velos enabled with HCD FT detection permits the rapid identification and relative quantitation of protein abundance as a result of stress-activated gene expression changes.
Development of a GC-EI/CI-enabled LTQ-orbitrap mass spectrometer for high mass accuracy and resolution GC-MS
Conventional GC/MS instruments employ ultrafast (often 20-50 Hz) and sensitive, but low to moderate resolution and mass accuracy mass analyzers: typically, the Paul-type or linear ion trap, the single quadrupole, or time-of-flight analyzer. Combined with internal retention time and mass calibration standards, the high throughput MS detector is an extremely effective tool for efficient determination of the presence and quantity of one or several well studied, known components in a matrix. If the targets of the GC/MS assay are truly unknown and multitudinous, or require resolution of fine isotopic structure, however, retention time and unit mass spectral data alone are insufficient to yield unambiguous elemental or structural identifications.
High mass accuracy and resolution analyzers, like the double-focusing magnetic sector and FT-ICR, are not new to the GC-MS community. The most well-established of these and one of the first to be coupled to GC, the magnetic sector instrument, is currently widely employed for trace detection of toxic tetra- through octa-substituted chlorodibenzo-p-dioxins and furans congeners under EPA method 1613. GC/FT-ICR MS was first reported on in 1980 by Ledford and colleagues1 and developed extensively during that decade. Recently, the Solouki group has demonstrated the breadth of potential profiling applications of this instrument, from petroleomics to the determination of gas-phase basicities.
These instruments typically operate in selective ion monitoring mode at resolutions of ~30k with ±5 ppm mass measurement accuracy and ~1 s scan speeds. Because sensitivity of the magnetic sector falls off precipitously at high resolution, this instrument is not well-suited for full mass range profiling or discovery applications.
The advent of the orbitrap and high-field orbitrap mass analyzers has further expanded the range of high mass accuracy and resolution instruments available. These instruments, capable of sub-ppm mass accuracy with internal calibration, resolutions up to 150k and 600k (high-field), and isotopic distribution error typically <3-10%3, have been extensively developed for use as hybrid and standalone instruments for LC/MS and MALDI/MS. We have developed the first adaption of the orbitrap mass analyzer for GC/MS. Using our group’s implementation of electron transfer dissociation (ETD) on a quadrupole linear ion trap (QLT)-orbitrap hybrid mass spectrometer, in which a negative chemical ionization (NCI) source was coupled distally at the rear lens of the c-trap via a long transfer octopole, as a template, we modified the NCI source to additionally permit electron ionization (EI) and positive chemical ionization (PCI), and coupled a gas chromatograph via a heated transfer line directly to the ionization region of the source.
Development of a high-throughput assay for screening potential ETD reagents using an ETD-enabled QLT-orbitrap coupled to a gas chromatograph
In all ion/ion reactions, electron transfer, proton transfer, and ion attachment are competitive processes. For the polyprotonated peptides typical of ETD-based proteomics applications, the choice of anionic reagent is crucial for efficient reaction by electron transfer pathways. As described by Gunawardena, the best anionic reagents feature low electron affinity and favorable Franck-Condon factors. While fluoranthene has become the reagent of choice for many ETD applications, better reagent anions have been described. Owing to the time-intensive nature of testing potential ETD reagents, however, there have been few reports profiling such reagents at length, none exhaustive. To this aim, we have developed a high-throughput assay for screening potential ETD reagents using an ETD-enabled quadrupole linear ion trap (QLT)-orbitrap hybrid mass spectrometer coupled to a gas chromatograph (GC).
An ETD-enabled QLT-orbitrap (Thermo Fisher Scientific, Bremen, Germany) was modified by coupling a GC via heated transfer line to the distal EI/CI source of the hybrid instrument. New instrument firmware was written to facilitate the following scan sequence: (1) GC analytes are ionized by methane negative chemical ionization (NCI) in the EI/CI source, (2) the resulting anion mixture is injected into the orbitrap for MS1 m/z analysis, (3) a candidate anion is injected into the QLT and isolated by automated data-dependent selection, (4) an exemplary peptide cation is injected into the QLT and isolated, (5) the reagent anion and cation precursor are reacted and (6) positive product ions are m/z analyzed in the QLT (MS2). In our preliminary proof-of-principle implementation, reactions were performed using triply charged substance P and a test bed of 16 polycyclic aromatic hydrocarbons (PAH).
Within a single one-hour gas chromatography run, we acquired thousands of MS1 full-scan anion and MS2 ion/ion reaction spectra. Using the high resolution and mass accuracy orbitrap MS1 scans preceding and following each ion/ion MS2 spectrum, intact mass measurements for the reagent anions confirmed the identity of all molecules in the PAH mixture, including naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenz(a,h)anthracene, and benzo(g,h,i)perylene. Each ion/ion spectrum was annotated and the reacting anion was assessed for its suitability as an ETD reagent. Our proof-of-principle implementation indicates the utility and feasibility of this assay for screening numerous potential ETD reagent anions in a high-throughput, ‘online’ manner.
Elucidation of the cellular signaling pathways which regulate human embryonic stem cell pluripotency and differentiation
Human embryonic stem (hES) cells hold great medicinal promise due to their ability to indefinitely self-renew and to differentiate into any type of cell in the adult human body. The understanding of cellular signaling events which regulate these unique cell states are crucial to harnessing this potential. In the Coon Lab, we use state-of-the-art mass spectrometry techniques to quantitatively probe the signaling mechanism of reversible protein phosphorylation in human ES cells. Identifying the proteins and phosphorylation site which change in response to treatments with different growing factors, morphogens, or expression patterns, provides the stem cell community with innovative knowledge to continue to push the boundaries of biology.
Novel IR photon-based strategies for peptide fragmentation
This projects aims to develop a collection of IR photon-based fragmentation techniques. These techniques include, but are not limited to, the use of IR photons for both direct peptide fragmentation (IRMPD) and the disruption of peptide gas phase secondary structure prior to ETD reactions (AI-ETD).
Instruments
Le Deuce
Thermo LTQ Orbitrap Velos:
Dual-cell quadrupole linear ion trap-Orbitrap hybrid MS.
Rooster
Thermo LTQ Orbitrap Velos ETD:
Dual-cell quadrupole linear ion trap-Orbitrap hybrid MS with NCI source for ETD.
Charger
Thermo LTQ Orbitrap Velos ETD:
Dual-cell quadrupole linear ion trap-Orbitrap hybrid MS with NCI source for ETD.
Cyclops
Thermo LTQ Velos ETD:
Dual-cell quadrupole linear ion trap MS with NCI source for ETD.
Infrared CO2 Laser
Synrad 10.6 nm infrared laser for enabling IR-based dissociation types (IRMPD, AI-ETD) on both standalone and hybrid mass spectrometers
Kenny
Thermo LTQ XL ETD:
Quadrupole linear ion trap MS with prototype NCI source for ETD.
Waters nanoACQUITY UPLC
Ultra performance high-pressure nano-scale liquid chromatograph (5 units)
Monty
Thermo TSQ Discovery Quantum:
Triple quadrupole MS
FinniganMAT GC
Gas chromatograph for GC/Orbitrap MS
Agilent 1100
High pressure liquid chromatograph
Advion TriVersa NanoMate
Integrated microchip-based nanoelectrospray robot for stable sample infusion
Thermo Surveyor HPLCs
High pressure liquid chromatographs for preparative-scale offline strong cation exchange and reversed-phase fractionation (3 units)
Wet Bench
Wet chemical bench space for mass spectrometry sample preparation. Includes a 37C incubator, cooled and uncooled centrifuges, analytical balances, 4, -20, and -80C freezers, pH meter, plate stirrer, etc.
Laser Puller
Sutter P-2000 Laser Based Micropipette Puller for fabrication of emitter tips for nano-flow electrospray ionization
Pressure Bomb
Pressure bombs for fabrication of nanocapillary columns for LC/MS
Funding
- The Beckman Foundation
- Eli Lilly and Company
- The National Institues of Health
- The National Science Foundation
- Thermo Fisher Scientific