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Permanent link for this collectionhttps://hdl.handle.net/2022/17214
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Item Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients(American Journal of Transplantation, 2017) Toby, Timothy K.; Kim, Kyunggon; Thomas, Paul M.; Fellers, Ryan T.; LeDuc, Richard D.; Kelleher, Neil L.; Demetris, Anthony J.; Abecassis, Michael M.; Levitsky, Josh M.Biomarker profiles diagnostic of acute rejection (AR) in liver transplant (LT) recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform-resolved signatures for AR in PBMC, using a novel top down proteomics methodology. We processed viable and non-viable PBMC lysates from 24 non-HCV+ LT recipients (age 60±15.6 years, 66.6% male, mean 3.7±3.3 year from LT) undergoing liver biopsy by molecular weight based fractionation and analyzed them by top-down proteomics in 3 steps: 1) Nanocapillary liquid chromatography coupled with high-resolution tandem mass spectrometry, 2) database searching to identify and characterize proteoforms, 3) data processing through a linear hierarchical model matching the study design to quantify proteoform fold changes in AR vs. Tx (normal liver function) vs. ADNR (acute dysfunction without rejection). Differentially abundant proteoforms were seen in patients with AR vs. TX and AR vs. ADNR, suggesting a distinct proteoform signature of AR in LT recipients. This was most evident in the viable cell preparations. Mapping analysis of these proteins back to genes through PANTHER and GO revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these signatures and test their predictive value for use in clinical management.Item A comprehensive pipeline for translational top-down proteomics from a single blood draw(Nature Protocols, 2016-11) Toby, Timothy K.; Fornelli, Luca; Srzentić, Kristina; DeHart, Caroline J.; Levitsky, Josh; Friedewald, John; Kelleher, Neil L.Top-down proteomics (TDP) by mass spectrometry (MS) has become increasingly popular in translational spheres as researchers discern the value of proteoform-resolved measurements. The advent of robust prefractionation strategies for intact proteoforms and the emerging availability of high-resolution benchtop mass spectrometers have catalyzed the emergence of high-throughput TDP for the analysis of biological and clinical samples. Here, we provide a comprehensive protocol for TDP of a sample type often investigated in translational research: human peripheral blood mononuclear cells (PBMC). The workflow described herein comprises sample collection, cell lysis, proteoform prefractionation, instrument setup, liquid chromatography-tandem MS (LC-MS/MS) analysis, and data processing, for a minimum completion time of 48 hours. Importantly, this workflow is compatible with multiple PBMC processing strategies and existing biorepository practices, can be used for both small-scale targeted proteoform studies or large-scale multi-sample discovery studies, and has been successfully applied in quantitative contexts for biomarker discovery.Item Identification and characterization of human proteoforms by top-down LC-21 tesla FT-ICR mass spectrometry(American Chemical Society, 2016) Anderson, Lissa C; DeHart, Caroline J; Kaiser, Nathan K; Fellers, Ryan T; Smith, Donald F; Greer, Joseph B; LeDuc, Richard D; Blackney, Gregory T; Thomas, Paul M; Kelleher, Neil L; Hendrickson, Christopher LThe National High Magnetic Field Laboratory (NHMFL) recently installed a 21T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here, we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identify a combined total of 684 unique protein entries observed as 3,238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from 40 LC-MS/MS experiments.Item Advancing top-down analysis of the human proteome using a benchtop quadrupole-Orbitrap mass spectrometer(Forthcoming, 2016-08) Fornelli, Luca; Durbin, Kenneth R; Fellers, Ryan T; Early, Bryan P; Greer, Joseph B; LeDuc, Richard D; Compton, Phillip D; Kelleher, Neil LOver the past decade, developments in high resolution mass spectrometry have enabled the high throughput analysis of intact proteins from complex proteomes, leading to the identification of thousands of proteoforms. Traditionally, top-down proteomics experiments relied on hybrid ion trap – Fourier transform mass spectrometers combined with data-dependent acquisition strategies. Here, we used a benchtop quadrupole – Orbitrap instrument coupled with unique data acquisition software to identify and characterize nearly 2,000 proteoforms at 1% false discovery rate from human fibroblasts.Item Accurate Estimation of False Discovery Rates for Protein and Proteoform Identification in Top Down Proteomics(Forthcoming) Shams, Daniel P; Early, Bryan P; Fellers, Ryan T; Greer, Joseph B; Thomas, Paul M; Fornelli, Luca; LeDuc, Richard D; Schwab, David J; Kelleher, Neil LWithin the last five years, top down proteomics (TDP) has emerged as a high throughput technique for protein identification in addition to characterization and quantitation of thousands of modified proteoforms. Here, a framework for calculating an accurate false discovery rate (FDR) that considers both protein and proteoform levels was used to evaluate local dependencies when aggregating results from replicate LC-MS/MS runs searched with different search modes and parameters.Ê We find that proteoform identifications are not statistically independent of each other and that correcting the FDR locally within a given LC-MS/MS run is not sufficient to control FDR globally across a large experiment.Ê A series of corrections used previously in genomics was implemented to address these issues and produce a global FDR calculation that scales well. ÊThe validity of the system is assessed by analyzing two previously published experimental datasets.ÊWeb-based access via a new TDPORTAL to high-performance computation enables all steps necessary to create a set of results utilizing the accurate and scalable FDR estimation described here. Also, a new application called TOP DOWN VIEWER enables viewing, analyzing, and sharing result sets via .tdReport files and is available at http://topdownviewer.northwestern.edu.Item Bioinformatics Analysis of Top-Down Mass Spectrometry Data(Methods in Molecular Biology, Springer) DeHart, Caroline J; Fellers, Ryan T; Fornelli, Luca; Kelleher, Neil L; Thomas, Paul MTraditional bottom-up mass spectrometry-based proteomics relies on the use of an enzyme, often trypsin, to generate small peptides (typically < 25 amino acids long). In top-down proteomics, proteins remain intact and are directly measured within the mass spectrometer. This technique, while inherently simpler than bottom-up proteomics, generates data which must be processed and analyzed using software tools “purpose-built” for the job. In this chapter, we will show the analysis of proteins from deconvolution and deisotoping through analysis with ProSight Lite, a free, vendor agnostic tool for the analysis of top-down mass spectrometry data. We will illustrate with two examples of intact protein spectra and discuss the iterative use of the software to characterize proteoforms and to discover the sites of post-translational modifications.Item Bioinformatics Analysis of Top-Down Mass Spectrometry Data with ProSight Lite(Methods in Molecular Biology, Springer, 2016) De Hart, Caroline J; Fellers, Ryan T; Fornelli, Luca; Kelleher, Neil L; Thomas, Paul MTraditional bottom-up mass spectrometry-based proteomics relies on the use of an enzyme, often trypsin, to generate small peptides (typically < 25 amino acids long). In top-down proteomics, proteins remain intact and are directly measured within the mass spectrometer. This technique, while inherently simpler than bottom-up proteomics, generates data which must be processed and analyzed using software tools “purpose-built” for the job. In this chapter, we will show the analysis of proteins from deconvolution and deisotoping through analysis with ProSight Lite, a free, vendor agnostic tool for the analysis of top-down mass spectrometry data. We will illustrate with two examples of intact protein spectra and discuss the iterative use of the software to characterize proteoforms and to discover the sites of post-translational modifications.Item Comparative top down proteomics of peripheral blood mononuclear cells from kidney transplant recipients with normal kidney biopsies or acute rejection(Proteomics, 2016-02-19) Savaryn, John P.; Toby, Timothy K.; Catherman, Adam D.; Fellers, Ryan T.; LeDuc, Richard D.; Thomas, Paul M.; Friedewald, John J.; Salomon, Daniel R.; Abecassis, Michael M.; Kelleher, Neil L.Recent studies utilizing transcriptomics, metabolomics, and bottom up proteomics have identified molecular signatures of kidney allograft pathology. Although these results make significant progress toward non-invasive differential diagnostics of dysfunction of a transplanted kidney, they provide little information on the intact, often modified, protein molecules present during progression of this pathology. Because intact proteins underpin diverse biological processes, measuring the relative abundance of their modified forms promises to advance mechanistic understanding, and might provide a new class of biomarker candidates. Here, we used top down proteomics to inventory the modified forms of whole proteins in peripheral blood mononuclear cells (PBMCs) taken at the time of kidney biopsy for 40 kidney allograft recipients either with healthy transplants or those suffering acute rejection. Supported by gas-phase fragmentation of whole protein ions during tandem mass spectrometry, we identified 344 proteins mapping to 2,905 distinct molecular forms (proteoforms). Using an initial implementation of a label-free approach to quantitative top down proteomics, we obtained evidence suggesting relative abundance changes in 111 proteoforms between the two patient groups. Collectively, our work is the first to catalog intact protein molecules in PBMCs and suggests differentially abundant proteoforms for further analysis.Item An informatic framework for decoding protein complexes by top-down mass spectrometry(Nature Publishing Group) Skinner, Owen S.; Havugimana, Pierre C.; Haverland, Nicole A.; Fornelli, Luca; Early, Bryan P.; Greer, Joseph B.; Fellers, Ryan T.; Durbin, Kenneth R.; Do Vale, Luis H. F.; Melani, Rafael D.; Seckler, Henrique S.; Nelp, Micah T.; Belov, Mikhail E.; Horning, Stevan R.; Makarov, Alexander A.; LeDuc, Richard D.; Bandarian, Vahe; Compton, Philip D.; Kelleher, Neil L.Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy for hierarchical top-down analysis, identification, and scoring of multi-proteoform complexes by native mass spectrometry.Item Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry(Journal of Proteomics, 2015-05-04) Savaryn, John Paul; Skinner, Owen S.; Fornelli, Luca; Fellers, Ryan T.; Compton, Philip D.; Terhune, Scott S.; Abecassis, Mike M.; Kelleher, Neil L.Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-?B p65 proteoforms isolated from human cells. We show isotopic resolution of N-terminally acetylated p65 and determined it is the most abundant proteoform expressed following transfection in 293T cells. We also show MS1 evidence for monophosphorylation of p65 under similar culture conditions and describe a high propensity for p65 proteoforms to fragment internally during beam-style MS2 fragmentation; up to 71% of the fragment ions could be matched as internals in some fragmentation spectra. Finally, we used native spray mass spectrometry to measure proteins copurifying with p65 and present evidence for the native detection of p65, 71 kDa heat shock protein, and p65 homodimer. Collectively, our work demonstrates the efficient isolation and top down mass spectrometry analysis of p65 from human cells, and we discuss the perturbations of overexpressing tagged proteins to study their biochemistry.Item Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry(Scienctific Reports, 2014-07-23) Kellie, JF; Higgs, RE; Ryder, JW; Major, A; Beach, TG; Adler, CH; Merchant, K; Knierman, MDA robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).Item Characterization of Native Protein Complexes Using Ultraviolet Photodissociation Mass SpectrometryO’Brien, John P.; Li, Wenzong; Zhang, Yan; Brodbelt, Jennifer S.Ultraviolet photodissociation (UVPD) mass spectrometry (MS) was used to characterize the sequences of proteins in native protein-ligand and protein-protein complexes and to provide auxiliary information about the binding sites of the ligands and protein-protein interfaces. UVPD outperformed collisional induced dissociation (CID) and higher-energy collisional dissociation (HCD) in terms of yielding the most comprehensive diagnostic primary sequence information of the proteins in the complexes. UVPD also generated non-covalent fragment ions containing a portion of the protein still bound to the ligand which revealed some insight into the nature of the binding sites of myoglobin/heme, eIF4E/m7GTP, and as well as human peptidyl-prolyl cis-trans isomerase Pin1 in complex with the peptide derived from the C-terminal domain of RNA polymerase II. Non-covalently bound protein-protein fragment ions from oligomeric beta-lactoglobulin dimers and hexameric insulin complexes were also produced upon UVPD, providing some illumination of tertiary and quaternary protein structural features.Item Ultraviolet Photodissociation for Characterization of Whole Proteins on a Chromatographic Time Scale.(2014-02-21) Cannon, JR; Cammarata, MB; Robotham, SA; Cotham, VC; Shaw, JB; Fellers, RT; Early, BP; Thomas, PM; Kelleher, NL; Brodbelt, JSIntact protein characterization using mass spectrometry thus far has been achieved at the cost of throughput. Presented here is the application of 193 nm ultraviolet photodissociation (UVPD) for top down identification and characterization of proteins in complex mixtures in an online fashion. Liquid chromatographic separation at the intact protein level coupled with fast UVPD and high-resolution detection resulted in confident identification of 46 unique sequences compared to 44 using HCD from prepared Escherichia coli ribosomes. Importantly, nearly all proteins identified in both the UVPD and optimized HCD analyses demonstrated a substantial increase in confidence in identification (as defined by an average decrease in E value of ∼40 orders of magnitude) due to the higher number of matched fragment ions. Also shown is the potential for high-throughput characterization of intact proteins via liquid chromatography (LC)−UVPD-MS of molecular weight-based fractions of a Saccharomyces cerevisiae lysate. In total, protein products from 215 genes were identified and found in 292 distinct proteoforms, 168 of which contained some type of post-translational modification.Item Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins.(Wiley VCH (Proteomics), 2014-01-14) Li, Yihan; Compton, Philip D.; Tran, John C.; Ntai, Ioanna; Kelleher, Neil L.The direct analysis of intact proteins via mass spectrometry offers compelling advantages in comparison to alternative methods due to the direct and unambiguous identification and characterization of protein sequences it provides. The inability to efficiently analyze proteins in the ‘middle mass range’, defined here as proteins from 30-80 kDa, in a robust fashion has limited the adoption of these “top-down” methods. Largely a result of poor liquid chromatographic performance, the limitations in this mass range may be addressed by alternative separations that replace chromatography. Herein, the short migration times of capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) have been extended to size-sorted whole proteins in complex mixtures from Pseudomonas aeruginosa PA01. An electrokinetically pumped nanospray interface, a coated capillary and a stacking method for on-column sample concentration were developed to achieve high loading capacity and separation resolution. We achieved full width at half maximum of 8-16 seconds for model proteins up to 29 kDa and identified 30 proteins in the mass range of 30-80 kDa from Pseudomonas aeruginosa PA01 whole cell lysate. These results suggest that CZE-ESI-MS/MS is capable of identifying proteins in the middle mass range in top-down proteomics.Item Autopilot: An Online Data Acquisition Control System for the Enhanced High-throughput Characterization of Intact Proteins(2014) Durbin, Kenneth; Fellers, Ryan; Ioanna, Ntai; Kelleher, Neil; Compton, PhilipThe ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context.