Publications by authors named "Caroline J DeHart"

22 Publications

  • Page 1 of 1

Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain.

J Proteome Res 2021 09 11;20(9):4427-4434. Epub 2021 Aug 11.

NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States.

Previous work employing five SARS-CoV-2 spike protein receptor-binding domain (RBD) constructs, comprising versions originally developed by Mt. Sinai or the Ragon Institute and later optimized in-house, revealed potential heterogeneity which led to questions regarding variable seropositivity assay performance. Each construct was subjected to N-deglycosylation and subsequent intact mass analysis, revealing significant deviations from predicted theoretical mass for all five proteins. Complementary tandem MS/MS analysis revealed the presence of an additional pyroGlu residue on the N-termini of the two Mt. Sinai RBD constructs, as well as on the N-terminus of the full-length spike protein from which they were derived, thus explaining the observed mass shift and definitively establishing the spike protein N-terminal sequence. Moreover, the observed mass additions for the three Ragon Institute RBD constructs were identified as variable N-terminal cleavage points within the signal peptide sequence employed for recombinant expression. To resolve this issue and minimize heterogeneity for further seropositivity assay development, the best-performing RBD construct was further optimized to exhibit complete homogeneity, as determined by both intact mass and tandem MS/MS analysis. This new RBD construct has been validated for seropositivity assay performance, is available to the greater scientific community, and is recommended for use in future assay development.
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http://dx.doi.org/10.1021/acs.jproteome.1c00349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419861PMC
September 2021

Production and Membrane Binding of N-Terminally Acetylated, C-Terminally Farnesylated and Carboxymethylated KRAS4b.

Methods Mol Biol 2021 ;2262:105-116

NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.

Recombinant mammalian proteins are routinely produced in E. coli and thus lack post-translational modifications. KRAS4b is processed at both the N- and C-terminus, resulting in an acetylation of the N-terminus (at Thr, after aminopeptidase removal of the original N-term Met) and farnesylation/carboxymethylation of the C-terminal Cys (after proteolytic cleavage of the original C-terminal three amino acids, Val-Iso-Met). Processing of KRAS enables it to associate with the plasma membrane and fulfill its function in cell signaling. We describe here the production of recombinant KRAS4b from our modified baculovirus/insect cell expression system that accurately incorporates these in vivo modifications to allow experiments that anchor KRAS4b to membrane mimetics (e.g., nanodiscs and liposomes).
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http://dx.doi.org/10.1007/978-1-0716-1190-6_6DOI Listing
June 2021

Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry.

Methods Mol Biol 2021 ;2262:47-64

Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.

The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.
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http://dx.doi.org/10.1007/978-1-0716-1190-6_3DOI Listing
June 2021

Increased Single-Spectrum Top-Down Protein Sequence Coverage in Trapping Mass Spectrometers with Chimeric Ion Loading.

Anal Chem 2020 09 2;92(18):12193-12200. Epub 2020 Sep 2.

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310, United States.

Fourier transform mass spectrometers routinely provide high mass resolution, mass measurement accuracy, and mass spectral dynamic range. In this work, we utilize 21 T Fourier transform ion cyclotron resonance (FT-ICR) to analyze product ions derived from the application of multiple dissociation techniques and/or multiple precursor ions within a single transient acquisition. This ion loading technique, which we call, "chimeric ion loading", saves valuable acquisition time, decreases sample consumption, and improves top-down protein sequence coverage. In the analysis of MCF7 cell lysate, we show collision-induced dissociation (CID) and electron-transfer dissociation (ETD) on each precursor on a liquid chromatography-mass spectrometry (LC-MS) timescale and improve mean sequence coverage dramatically (CID-only 15% vs chimeric 33%), even during discovery-based acquisition. This approach can also be utilized to multiplex the acquisition of product ion spectra of multiple charge states from a single protein precursor or multiple ETD/proton-transfer reactions (PTR) reaction periods. The analytical utility of chimeric ion loading is demonstrated for top-down proteomics, but it is also likely to be impactful for tandem mass spectrometry applications in other areas.
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http://dx.doi.org/10.1021/acs.analchem.0c01064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845485PMC
September 2020

NMT1 and NMT2 are lysine myristoyltransferases regulating the ARF6 GTPase cycle.

Nat Commun 2020 02 26;11(1):1067. Epub 2020 Feb 26.

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.
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http://dx.doi.org/10.1038/s41467-020-14893-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044312PMC
February 2020

Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics.

Mol Cell Proteomics 2020 02 30;19(2):405-420. Epub 2019 Dec 30.

Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208. Electronic address:

Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.
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http://dx.doi.org/10.1074/mcp.TIR119.001638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000117PMC
February 2020

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

Nat Methods 2019 07 27;16(7):587-594. Epub 2019 Jun 27.

Barnett Institute of Chemical and Biological Analysis and Departments of Chemistry & Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, MA, USA.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.
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http://dx.doi.org/10.1038/s41592-019-0457-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719561PMC
July 2019

A comprehensive pipeline for translational top-down proteomics from a single blood draw.

Nat Protoc 2019 01;14(1):119-152

Departments of Chemistry and of Molecular Biosciences, Northwestern University, Evanston, IL, USA.

Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.
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http://dx.doi.org/10.1038/s41596-018-0085-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439476PMC
January 2019

Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria.

Nat Commun 2018 10 15;9(1):4276. Epub 2018 Oct 15.

Departments of Molecular Biosciences and Chemistry, Northwestern University, Evanston, 60208, IL, USA.

Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide. Here we show that PmoD, a protein encoded within many pmo operons that is homologous to the AmoD proteins encoded within AOB amo operons, forms a copper center that exhibits the features of a well-defined Cu site using a previously unobserved ligand set derived from a cupredoxin homodimer. PmoD is critical for copper-dependent growth on methane, and genetic analyses strongly support a role directly related to pMMO and AMO. These findings identify a copper-binding protein that may represent a missing link in the function of enzymes critical to the global carbon and nitrogen cycles.
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http://dx.doi.org/10.1038/s41467-018-06681-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189053PMC
October 2018

The unique biomineralization transcriptome and proteome of Lytechinus variegatus teeth.

Connect Tissue Res 2018 12;59(sup1):20-29

a Feinberg School of Medicine, Department of Cell and Molecular Biology , Northwestern University , Chicago , IL , USA.

Background: Matrix-regulated biomineralization involves the specific nucleation and growth of mineral phases within or upon preformed structured organic matrices. We hypothesized that there might be a general mechanism whereby anionic, phosphorylated mineral ion-binding proteins assist in specifically locating the mineral ions with respect to the mineralizing structural organic matrix. Here we extended these studies to invertebrate mineralization in Lytechinus variegatus (Lv) teeth.

Materials And Methods: The tooth proteins were extracted and the phosphoproteins occluded in the mineral were enriched by passage through a ProQ Diamond phosphoprotein enrichment column, and subjected to MS/MS analysis. A Lv RNA-seq derived transcriptome database was generated. The MS/MS data found 25 proteins previously classified as "Predicted uncharacterized proteins" and many of the spicule matrix proteins. As these 25 proteins were also identified with the transcriptome analysis, and were thus no longer "hypothetical" but real proteins in the Lv tooth. Each protein was analyzed for the presence of a signal peptide, an acidic pI≤4, and the ability to be phosphorylated.

Results: Four new Lv tooth specific Pro-Ala-rich proteins were found, representing a new class of proteins.

Conclusion: The tooth is different from the spicules and other urchin skeletal elements in that only the tooth contains both "high" and "very high" magnesium calcite, [Ca(1-X) Mg(X) CO3], where X is the mole fraction of Mg. We speculate that our newly discovered proline-alanine rich proteins, also containing sequences of acidic amino acids, may be involved in the formation of high magnesium and very high magnesium calcite.
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http://dx.doi.org/10.1080/03008207.2017.1408605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052879PMC
December 2018

Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk.

Proc Natl Acad Sci U S A 2018 04 2;115(16):4140-4145. Epub 2018 Apr 2.

Department of Chemistry, Proteomics Center of Excellence, Northwestern University, Evanston, IL 60208;

Mutations of the gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.
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http://dx.doi.org/10.1073/pnas.1716122115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910823PMC
April 2018

The biosynthesis of methanobactin.

Science 2018 Mar;359(6382):1411-1416

Department of Molecular Biosciences and Department of Chemistry, Northwestern University, Evanston, IL 60208, USA.

Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.
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http://dx.doi.org/10.1126/science.aap9437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944852PMC
March 2018

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer.

J Proteome Res 2017 05 19;16(5):2072-2079. Epub 2017 Apr 19.

Department of Chemistry, University of Texas at Austin , Austin, Texas 78712, United States.

The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatographic time scale. However, utilization of UVPD for high-throughput top-down analysis to date has been limited by bioinformatics. Here we detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissociation (HCD) in a comparative analysis of HeLa whole-cell lysate by qualitative top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores greater than 40 for UVPD and 63% for HCD. The average sequence coverage (28 ± 20% for UVPD versus 17 ± 8% for HCD, p < 0.0001) was found to be higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.
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http://dx.doi.org/10.1021/acs.jproteome.7b00043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549616PMC
May 2017

Top-down proteomics: Where we are, where we are going?

J Proteomics 2018 03 7;175:3-4. Epub 2017 Feb 7.

Departments of Chemistry and Molecular Biosciences, Northwestern University, 2170 Campus Drive, Evanston, IL 60208, United States. Electronic address:

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http://dx.doi.org/10.1016/j.jprot.2017.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547015PMC
March 2018

Bioinformatics Analysis of Top-Down Mass Spectrometry Data with ProSight Lite.

Methods Mol Biol 2017 ;1558:381-394

Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA.

Traditional 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 intact protein spectra through deconvolution, deisotoping, and searching 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 fragmentation spectra and discuss the iterative use of the software to characterize proteoforms and discover the sites of post-translational modifications.
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http://dx.doi.org/10.1007/978-1-4939-6783-4_18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373093PMC
February 2018

Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein.

J Proteome Res 2017 02 23;16(2):920-932. Epub 2017 Jan 23.

Department of Biological Sciences, North Carolina State University , Raleigh, North Carolina 27695, United States.

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.
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http://dx.doi.org/10.1021/acs.jproteome.6b00873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5401637PMC
February 2017

Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry.

J Proteome Res 2017 02 12;16(2):1087-1096. Epub 2016 Dec 12.

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , Tallahassee, Florida 32310, United States.

Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T 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 identified a combined total of 684 unique protein entries observed as 3238 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 just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.
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http://dx.doi.org/10.1021/acs.jproteome.6b00696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353847PMC
February 2017

Bone protein "extractomics": comparing the efficiency of bone protein extractions of in tandem mass spectrometry, with an eye towards paleoproteomics.

PeerJ 2016 27;4:e2603. Epub 2016 Oct 27.

Proteomics Center of Excellence and Departments of Chemistry, Molecular Biosciences, and the Feinberg School of Medicine, Northwestern University , Evanston , IL , United States.

Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different "extractomes" will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils.
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http://dx.doi.org/10.7717/peerj.2603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088622PMC
October 2016

Characterization of Methanobactin from Methylosinus sp. LW4.

J Am Chem Soc 2016 09 26;138(35):11124-7. Epub 2016 Aug 26.

Departments of †Molecular Biosciences and ‡Chemistry, Northwestern University , Evanston, Illinois 60208, United States.

Methanobactins (Mbns) are a growing family of ribosomally produced, post-translationally modified natural products. Characteristic nitrogen-containing heterocycles and neighboring thioamides allow these compounds to bind copper with high affinity. Genome mining has enabled the identification of Mbn operons in bacterial genomes and the prediction of diverse Mbn structures from operon content and precursor peptide sequence. Here we report the characterization of Mbn from Methylosinus (Ms.) species (sp.) LW4. The peptide backbone is distinct from all previously characterized Mbns, and the post-translational modifications correspond precisely to those predicted on the basis of the Ms. sp. LW4 Mbn operon. Thus, prediction based on genome analysis combined with isolation and structural characterization represents a phylogenetic approach to finding diverse Mbns and elucidating their biosynthetic pathways.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074052PMC
http://dx.doi.org/10.1021/jacs.6b06821DOI Listing
September 2016

Impact of the adenoviral E4 Orf3 protein on the activity and posttranslational modification of p53.

J Virol 2015 Mar 7;89(6):3209-20. Epub 2015 Jan 7.

Princeton University, Department of Molecular Biology, Lewis Thomas Laboratory, Princeton, New Jersey, USA

Unlabelled: Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1-17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076-1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells.

Importance: The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3 ubiquitin ligase that contains the viral E1B 55-kDa and E4 Orf6 proteins, while the E4 Orf3 protein has been reported to block its ability to stimulate expression of p53-dependent genes. The comparisons reported here of the posttranslational modifications and activities of p53 populations that accumulate in infected normal human cells in the absence of both mechanisms of inactivation or of only the E3 ligase revealed little impact of the E4 Orf3 protein. These observations indicate that E4 Orf3-dependent disruption of Pml bodies does not have a major effect on the pattern of p53 posttranslational modifications in adenovirus-infected cells. Furthermore, they suggest that one or more additional viral proteins contribute to blocking p53 activation and the consequences that are deleterious for viral reproduction, such as apoptosis or cell cycle arrest.
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http://dx.doi.org/10.1128/JVI.03072-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337557PMC
March 2015

Extensive post-translational modification of active and inactivated forms of endogenous p53.

Mol Cell Proteomics 2014 Jan 20;13(1):1-17. Epub 2013 Sep 20.

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544.

The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues-for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated.
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http://dx.doi.org/10.1074/mcp.M113.030254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879606PMC
January 2014

The repression domain of the E1B 55-kilodalton protein participates in countering interferon-induced inhibition of adenovirus replication.

J Virol 2013 Apr 6;87(8):4432-44. Epub 2013 Feb 6.

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey, USA.

To begin to investigate the mechanism by which the human adenovirus type 5 E1B 55-kDa protein protects against the antiviral effects of type 1 interferon (IFN) (J. S. Chahal, J. Qi, and S. J. Flint, PLoS Pathog. 8:e1002853, 2012 [doi:10.1371/journal.ppat.1002853]), we examined the effects of precise amino acid substitution in this protein on resistance of viral replication to the cytokine. Only substitution of residues 443 to 448 of E1B for alanine (E1B Sub19) specifically impaired production of progeny virus and resulted in a large defect in viral DNA synthesis in IFN-treated normal human fibroblasts. Untreated or IFN-treated cells infected by this mutant virus (AdEasyE1Sub19) contained much higher steady-state concentrations of IFN-inducible GBP1 and IFIT2 mRNAs than did wild-type-infected cells and of the corresponding newly transcribed pre-mRNAs, isolated exploiting 5'-ethynyluridine labeling and click chemistry. These results indicated that the mutations created by substitution of residues 443 to 448 for alanine (Sub19) impair repression of transcription of IFN-inducible genes, by the E1B, 55-kDa protein, consistent with their location in a segment required for repression of p53-dependent transcription. However, when synthesized alone, the E1B 55-kDa protein inhibited expression of the p53-regulated genes BAX and MDM2 but had no impact whatsoever on induction of IFIT2 and GBP1 expression by IFN. These observations correlate repression of transcription of IFN-inducible genes by the E1B 55-kDa protein with protection against inhibition of viral genome replication and indicate that the E1B 55-kDa protein is not sufficient to establish such transcriptional repression.
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http://dx.doi.org/10.1128/JVI.03387-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624377PMC
April 2013
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