Publications by authors named "David R Goodlett"

234 Publications

Interfaces with Structure Dynamics of the Workhorses from Cells Revealed through Cross-Linking Mass Spectrometry (CLMS).

Biomolecules 2021 Mar 4;11(3). Epub 2021 Mar 4.

International Centre for Cancer Vaccine Science, University of Gdansk, ul. Kładki 24, 80-822 Gdansk, Poland.

The fundamentals of how protein-protein/RNA/DNA interactions influence the structures and functions of the workhorses from the cells have been well documented in the 20th century. A diverse set of methods exist to determine such interactions between different components, particularly, the mass spectrometry (MS) methods, with its advanced instrumentation, has become a significant approach to analyze a diverse range of biomolecules, as well as bring insights to their biomolecular processes. This review highlights the principal role of chemistry in MS-based structural proteomics approaches, with a particular focus on the chemical cross-linking of protein-protein/DNA/RNA complexes. In addition, we discuss different methods to prepare the cross-linked samples for MS analysis and tools to identify cross-linked peptides. Cross-linking mass spectrometry (CLMS) holds promise to identify interaction sites in larger and more complex biological systems. The typical CLMS workflow allows for the measurement of the proximity in three-dimensional space of amino acids, identifying proteins in direct contact with DNA or RNA, and it provides information on the folds of proteins as well as their topology in the complexes. Principal CLMS applications, its notable successes, as well as common pipelines that bridge proteomics, molecular biology, structural systems biology, and interactomics are outlined.
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http://dx.doi.org/10.3390/biom11030382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001575PMC
March 2021

Deep-sea microbes as tools to refine the rules of innate immune pattern recognition.

Sci Immunol 2021 Mar;6(57)

Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA.

The assumption of near-universal bacterial detection by pattern recognition receptors is a foundation of immunology. The limits of this pattern recognition concept, however, remain undefined. As a test of this hypothesis, we determined whether mammalian cells can recognize bacteria that they have never had the natural opportunity to encounter. These bacteria were cultivated from the deep Pacific Ocean, where the genus was identified as a common constituent of the culturable microbiota. Most deep-sea bacteria contained cell wall lipopolysaccharide (LPS) structures that were expected to be immunostimulatory, and some deep-sea bacteria activated inflammatory responses from mammalian LPS receptors. However, LPS receptors were unable to detect 80% of deep-sea bacteria examined, with LPS acyl chain length being identified as a potential determinant of immunosilence. The inability of immune receptors to detect most bacteria from a different ecosystem suggests that pattern recognition strategies may be defined locally, not globally.
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http://dx.doi.org/10.1126/sciimmunol.abe0531DOI Listing
March 2021

An Update on MRMAssayDB: A Comprehensive Resource for Targeted Proteomics Assays in the Community.

J Proteome Res 2021 Apr 8;20(4):2105-2115. Epub 2021 Mar 8.

University of Victoria - Genome BC Proteomics Centre, Victoria, British Columbia V8Z 7X8, Canada.

Precise multiplexed quantification of proteins in biological samples can be achieved by targeted proteomics using multiple or parallel reaction monitoring (MRM/PRM). Combined with internal standards, the method achieves very good repeatability and reproducibility enabling excellent protein quantification and allowing longitudinal and cohort studies. A laborious part of performing such experiments lies in the preparation steps dedicated to the development and validation of individual protein assays. Several public repositories host information on targeted proteomics assays, including NCI's Clinical Proteomic Tumor Analysis Consortium assay portals, PeptideAtlas SRM Experiment Library, SRMAtlas, PanoramaWeb, and PeptideTracker, with all offering varying levels of details. We introduced MRMAssayDB in 2018 as an integrated resource for targeted proteomics assays. The Web-based application maps and links the assays from the repositories, includes comprehensive up-to-date protein and sequence annotations, and provides multiple visualization options on the peptide and protein level. We have extended MRMAssayDB with more assays and extensive annotations. Currently it contains >828 000 assays covering >51 000 proteins from 94 organisms, of which >17 000 proteins are present in >2400 biological pathways, and >48 000 mapping to >21 000 Gene Ontology terms. This is an increase of about four times the number of assays since introduction. We have expanded annotations of interaction, biological pathways, and disease associations. A newly added visualization module for coupled molecular structural annotation browsing allows the user to interactively examine peptide sequence and any known PTMs and disease mutations, and map all to available protein 3D structures. Because of its integrative approach, MRMAssayDB enables a holistic view of suitable proteotypic peptides and commonly used transitions in empirical data. Availability: http://mrmassaydb.proteincentre.com.
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http://dx.doi.org/10.1021/acs.jproteome.0c00961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041396PMC
April 2021

MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.

J Proteome Res 2021 Jan 25. Epub 2021 Jan 25.

University of Gdansk, International Centre for Cancer Vaccine Science, ul. Kładki 24, 80-822 Gdansk, Poland.

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.
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http://dx.doi.org/10.1021/acs.jproteome.0c00629DOI Listing
January 2021

Proteomic profile of vitreous in patients with tubercular uveitis.

Tuberculosis (Edinb) 2021 01 3;126:102036. Epub 2020 Dec 3.

Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India. Electronic address:

Objective: To elucidate disease-specific host protein profile in vitreous fluid of patients with intraocular inflammation due to tubercular uveitis (TBU).

Methods: Vitreous samples from 13 patients with TBU (group A), 7 with non-TBU (group B) and 9 with no uveitis (group C) were analysed by shotgun proteomics using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were subjected to pathway analysis using WEB-based Gene SeT Analysis Toolkit software.

Results: Compared to control groups (B + C combined), group A (TBU) displayed 32 (11 upregulated, 21 downregulated) DEPs, which revealed an upregulation of coagulation cascades, complement and classic pathways, and downregulation of metabolism of carbohydrates, gluconeogenesis, glucose metabolism and glycolysis/gluconeogenesis pathways. When compared to group B (non-TBU) alone, TBU displayed 58 DEPs (21 upregulated, 37 downregulated), with an upregulation of apoptosis, KRAS signaling, diabetes pathways, classic pathways, and downregulation of MTORC1 signaling, glycolysis/gluconeogenesis, and glucose metabolism.

Conclusion: This differential protein profile provides novel insights into the molecular mechanisms of TBU and a baseline to explore vitreous biomarkers to differentiate TBU from non-TBU, warranting future studies to identify and validate them as a diagnostic tool in TBU. The enriched pathways generate interesting hypotheses and drive further research.
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http://dx.doi.org/10.1016/j.tube.2020.102036DOI Listing
January 2021

Rapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids.

Sci Rep 2020 12 9;10(1):21536. Epub 2020 Dec 9.

Pataigin, LLC, Seattle, WA, USA.

Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.
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http://dx.doi.org/10.1038/s41598-020-78401-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725828PMC
December 2020

Streamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI.

J Am Soc Mass Spectrom 2020 Dec 5;31(12):2495-2502. Epub 2020 Oct 5.

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore 21201, Maryland, United States.

Cardiolipins (CLs) are an important, regulated lipid class both in prokaryotic and eukaryotic cells, yet they remain largely unexplored by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in tissues. To date, no in-depth optimization studies of label-free visualization of CLs in complex biological samples have been reported. Here we report a streamlined modification to our previously reported MALDI-MSI method for detection of endogenous CLs in prokaryotic and eukaryotic cells based on preparation with norharmane (NRM) matrix. Notably, the use of NRM matrix permitted sensitive detection (4.7 pg/mm) of spotted CL synthetic standards. By contrast, four other MALDI matrices commonly used for lipid analysis failed to generate CL ions. Using this NRM-based method, endogenous CLs were detected from two types of complex biological samples: dried bacterial arrays and mouse tissue sections. In both cases, using NRM resulted in a better signal/noise for CL ions than the other matrices. Furthermore, inclusion of a washing step improved CL detection from tissue and this combined tissue preparation method (washing and NRM matrix) was used to profile normal mouse lung. Mouse lung yielded 26 unique CLs that were mapped and identified. Consistent with previous findings, CLs containing polyunsaturated fatty acids (PUFAs) were found in abundance in the airway and vascular features of the lung. This work represents a comprehensive investigation of detection conditions for CL using MALDI-MSI in complex biological samples that resulted in a streamlined method that enables future studies of the biological role(s) of CL in tissue.
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http://dx.doi.org/10.1021/jasms.0c00201DOI Listing
December 2020

On-Tissue Derivatization of Lipopolysaccharide for Detection of Lipid A Using MALDI-MSI.

Anal Chem 2020 10 28;92(20):13667-13671. Epub 2020 Sep 28.

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland 21201, United States.

We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host-pathogen interactions. We previously demonstrated detection of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host-pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, (, / 1797) and (, / 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and O-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major and lipid A species (/ 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host-pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host-pathogen interface.
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http://dx.doi.org/10.1021/acs.analchem.0c02566DOI Listing
October 2020

Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in .

Proc Natl Acad Sci U S A 2020 09 31;117(37):22984-22991. Epub 2020 Aug 31.

Department of Microbial Pathogenesis, University of Maryland, Baltimore, MD 21201;

Immune evasion through membrane remodeling is a hallmark of pathogenesis. remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens () and (), as well as the causative agent of plague, (). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory and strains, but not in immune-evasive Analysis of gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in and , lipid A isolated from a strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in , a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in infectivity, pathogenesis, and host innate immune evasion.
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http://dx.doi.org/10.1073/pnas.1917504117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502761PMC
September 2020

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Sep 19;31(9):1783-1802. Epub 2020 Aug 19.

The University of Edinburgh, EH9 3FJ Edinburgh, United Kingdom.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.
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http://dx.doi.org/10.1021/jasms.0c00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539639PMC
September 2020

Droplet delivery and nebulization system using surface acoustic wave for mass spectrometry.

Lab Chip 2020 08;20(17):3269-3277

School of Dentistry, University of Maryland, Baltimore, MD 21201, USA and International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland, EU.

We present a piezoelectric transducer for standing wave surface acoustic wave nebulization (SW-SAWN). The transducer nebulizes nonvolatile analytes present in bulk fluid into ambient air after which the aerosolized drops are sampled by mass spectrometry (MS) for detection. Furthermore, we report for the first time integration of anisotropic ratchet conveyors (ARCs) on the SAWN transducer surfaces to automate the sample preparation and droplet delivery process. The ARCs employ micro-sized hydrophilic patterns on hydrophobic Cytop coatings. Moving, positioning, merging, and mixing of droplets at a designated nebulization location are demonstrated. To create the ARCs, we adopt parylene C as a stencil mask so that the hydrophobicity of the Cytop does not degrade during the microfabrication process. MS measurements with the SAWN chip are performed under different input frequencies. The SAWN transducer can provide a controllable nebulization rate by varying the input nebulization frequency while maintaining a reasonable signal to noise ratio for MS detection.
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http://dx.doi.org/10.1039/d0lc00495bDOI Listing
August 2020

MGMS2: Membrane glycolipid mass spectrum simulator for polymicrobial samples.

Rapid Commun Mass Spectrom 2020 Aug;34(16):e8824

Department of Microbial Pathogenesis, University of Maryland, Baltimore, MD, USA.

Rationale: Polymicrobial samples present unique challenges for mass spectrometric identification. A recently developed glycolipid technology has the potential to accurately identify individual bacterial species from polymicrobial samples. In order to develop and validate bacterial identification algorithms (e.g. machine learning) using this glycolipid technology, generating a large number of various polymicrobial samples can be beneficial, but it is costly and labor-intensive. Here, we propose an alternative cost-effective approach that generates realistic in silico polymicrobial glycolipid mass spectra.

Methods: We introduce MGMS2 (membrane glycolipid mass spectrum simulator) as a simulation software package that generates in silico polymicrobial membrane glycolipid matrix-assisted laser desorption/ionization time-of-flight mass spectra. Unlike currently available simulation algorithms for polymicrobial mass spectra, the proposed algorithm considers errors in m/z values and variances of intensity values, occasions of missing signature ions, and noise peaks. To our knowledge, this is the first stand-alone bacterial membrane glycolipid mass spectral simulator. MGMS2 software and its manual are freely available as an R package. An interactive MGSM2 app that helps users explore various simulation parameter options is also available.

Results: We demonstrated the performance of MGSM2 using six microbes. The software generated in silico glycolipid mass spectra that are similar to real polymicrobial glycolipid mass spectra. The maximum correlation between in silico mass spectra generated by MGMS2 and the real polymicrobial mass spectrum was about 87%.

Conclusions: We anticipate that MGMS2, which considers spectrum-to-spectrum variation, will advance the bacterial algorithm development for polymicrobial samples.
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http://dx.doi.org/10.1002/rcm.8824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466374PMC
August 2020

Toll-like Receptor 4-Independent Effects of Lipopolysaccharide Identified Using Longitudinal Serum Proteomics.

J Proteome Res 2020 03 24;19(3):1258-1266. Epub 2020 Feb 24.

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland 21201, United States.

Sepsis remains one of the most lethal and costly conditions treated in U.S. hospitals, with approximately 50% of cases caused by Gram-negative bacterial infections. Septic shock is induced when lipopolysaccharide (LPS), the main component of Gram-negative outer bacterial membrane, signals through the Toll-like receptor 4 (TLR4) complex. Lethal endotoxemia, a model for septic shock, was induced in WT C57BL6 and TLR4 mice by administration of LPS. WT LPS treated mice showed high morbidity, while PBS treated LPS and treated TLR4 mice did not. ANOVA analysis of label-free quantification of longitudinal serum proteome revealed 182 out of 324 proteins in LPS injected WT mice that were significantly changed across four time points (0, 6, 12, and 18 h). No significant changes were identified in the two control groups. From the 182 identified proteins, examples of known sepsis biomarkers were validated by ELISA, which showed similar trends as MS proteomics data. Longitudinal analysis within individual mice produced 3-fold more significantly changed proteins than pair-wise comparison. A subsequent global analysis of WT and TLR4 mice identified pathways activated independent of TLR4. These pathways represent possible compensatory mechanisms that allow for control of Gram-negative bacterial infection regardless of host immune status.
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http://dx.doi.org/10.1021/acs.jproteome.9b00765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108756PMC
March 2020

The Role of TLRs in Anti-cancer Immunity and Tumor Rejection.

Front Immunol 2019 22;10:2388. Epub 2019 Oct 22.

International Centre for Cancer Vaccine Science (ICCVS), University of Gdaǹsk, Gdaǹsk, Poland.

In recent years, a lot of scientific interest has focused on cancer immunotherapy. Although chronic inflammation has been described as one of the hallmarks of cancer, acute inflammation can actually trigger the immune system to fight diseases, including cancer. Toll-like receptor (TLR) ligands have long been used as adjuvants for traditional vaccines and it seems they may also play a role enhancing efficiency of tumor immunotherapy. The aim of this perspective is to discuss the effects of TLR stimulation in cancer, expression of various TLRs in different types of tumors, and finally the role of TLRs in anti-cancer immunity and tumor rejection.
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http://dx.doi.org/10.3389/fimmu.2019.02388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817561PMC
November 2020

Proteome analysis of tissues by mass spectrometry.

Mass Spectrom Rev 2019 08 7;38(4-5):403-441. Epub 2019 Aug 7.

van 't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands.

Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm obtained with laser capture microdissection to much larger amounts that weight several milligrams.
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http://dx.doi.org/10.1002/mas.21598DOI Listing
August 2019

Model-Based Spectral Library Approach for Bacterial Identification via Membrane Glycolipids.

Anal Chem 2019 09 15;91(17):11482-11487. Epub 2019 Aug 15.

Department of Microbial Pathogenesis, School of Dentistry , University of Maryland , Baltimore , Maryland 21201 , United States.

By circumventing the need for a pure colony, MALDI-TOF mass spectrometry of bacterial membrane glycolipids (lipid A) has the potential to identify microbes more rapidly than protein-based methods. However, currently available bioinformatics algorithms (e.g., dot products) do not work well with glycolipid mass spectra such as those produced by lipid A, the membrane anchor of lipopolysaccharide. To address this issue, we propose a spectral library approach coupled with a machine learning technique to more accurately identify microbes. Here, we demonstrate the performance of the model-based spectral library approach for microbial identification using approximately a thousand mass spectra collected from multi-drug-resistant bacteria. At false discovery rates < 1%, our approach identified many more bacterial species than the existing approaches such as the Bruker Biotyper and characterized over 97% of their phenotypes accurately. As the diversity in our glycolipid mass spectral library increases, we anticipate that it will provide valuable information to more rapidly treat infected patients.
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http://dx.doi.org/10.1021/acs.analchem.9b03340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204536PMC
September 2019

Multi-Omics Strategies Uncover Host-Pathogen Interactions.

ACS Infect Dis 2019 04 25;5(4):493-505. Epub 2019 Mar 25.

Laboratory of Immune System Biology (LISB), National Institute of Allergy and Infectious Diseases , National Institutes of Health (NIH) , 9000 Rockville Pike , Bethesda , Maryland 20814 , United States.

With the success of the Human Genome Project, large-scale systemic projects became a reality that enabled rapid development of the systems biology field. Systems biology approaches to host-pathogen interactions have been instrumental in the discovery of some specifics of Gram-negative bacterial recognition, host signal transduction, and immune tolerance. However, further research, particularly using multi-omics approaches, is essential to untangle the genetic, immunologic, (post)transcriptional, (post)translational, and metabolic mechanisms underlying progression from infection to clearance of microbes. The key to understanding host-pathogen interactions lies in acquiring, analyzing, and modeling multimodal data obtained through integrative multi-omics experiments. In this article, we will discuss how multi-omics analyses are adding to our understanding of the molecular basis of host-pathogen interactions and systemic maladaptive immune response of the host to microbes and microbial products.
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http://dx.doi.org/10.1021/acsinfecdis.9b00080DOI Listing
April 2019

Temporal proteomic profiling reveals changes that support Burkholderia biofilms.

Pathog Dis 2019 03;77(2)

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA.

Melioidosis associated with opportunistic pathogen Burkholderia pseudomallei imparts a huge medical burden in Southeast Asia and Australia. At present there is no available human vaccine that protects against B. pseudomallei infection and antibiotic treatments are limited particularly for drug-resistant strains and bacteria in biofilm forms. Biofilm forming bacteria exhibit phenotypic features drastically different to their planktonic states, often exhibiting a diminished response to antimicrobial therapies. Our earlier work on global profiling of bacterial biofilms using transcriptomics and proteomics revealed transcript-decoupled protein abundance in bacterial biofilms. Here we employed reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to deduce temporal proteomic differences in planktonic and biofilm forms of Burkholderia thailandensis, which is weakly surrogate model of pathogenic B. pseudomallei as sharing a key element in genomic similarity. The proteomic analysis of B. thailandensis in biofilm versus planktonic states revealed that proteome changes support biofilm survival through decreased abundance of metabolic proteins while increased abundance of stress-related proteins. Interestingly, the protein abundance including for the transcription protein TEX, outer periplasmic TolB protein, and the exopolyphosphatase reveal adaption in bacterial biofilms that facilitate antibiotic tolerance through a non-specific mechanism. The present proteomics study of B. thailandensis biofilms provides a global snapshot of protein abundance differences and antimicrobial sensitivities in planktonic and sessile bacteria.
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http://dx.doi.org/10.1093/femspd/ftz005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482045PMC
March 2019

The effect of embryonic origin on the osteoinductive potential of bone allografts.

J Prosthet Dent 2019 Apr 28;121(4):651-658. Epub 2018 Dec 28.

Associate Professor, Department of Advanced Oral Sciences and Therapeutics, University of Maryland School of Dentistry, Baltimore, Md.

Statement Of Problem: Allografts with osteoinduction potential are widely used to augment bone in surgical and prosthetic rehabilitations. However, osteoinduction potential varies among commercially available allografts. Donor bones are derived from different embryonic origins, either the neural crest or mesoderm. Whether the origin of the bones affects the osteoinductivity of allograftsis is unclear.

Purpose: The purpose of this ex vivo study was to investigate the osteoinduction potential of allografts derived from bones with distinct embryonic origins.

Material And Methods: Allografts were obtained from human frontal and parietal bones at 2 different ages (fetal and adult). The specimens were divided into 4 groups: adult frontal (n=5), adult parietal (n=5), fetal frontal (n=10), and fetal parietal (n=10). Two investigations were conducted to assess the osteoinductive potential of these allografts. First, the osteogenesis of human osteoblasts exposed to these allografts were evaluated by analyzing the expression of runt-related transcription factor 2 (RUNX2), collagen type 1 alpha 2 chain (COL1A2), and bone gamma-carboxyglutamate protein (BGLAP) genes using quantitative real-time polymerase chain reaction (qRT-PCR). Second, the protein content of the adult frontal and parietal bone matrices was analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). One-way ANOVA and the t test were used for statistical analyses of the gene and protein expression of the groups (α=.05).

Results: No difference was found in the gene expression of the cells exposed to frontal or parietal bones. However, all 3 genes were significantly overexpressed in cells treated with fetal bones compared with adult bones. No difference was found in protein expression between adult frontal and adult parietal bones.

Conclusions: No difference was found in the osteoinductive capacity of frontal and parietal bones used as allografts. However, the osteoinductivity of fetal bones can be higher than that of adult bones. Further microanalyses are needed to determine the protein content of fetal bones.
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http://dx.doi.org/10.1016/j.prosdent.2018.09.003DOI Listing
April 2019

Rapid Microbial Identification and Antibiotic Resistance Detection by Mass Spectrometric Analysis of Membrane Lipids.

Anal Chem 2019 01 4;91(2):1286-1294. Epub 2019 Jan 4.

Department of Pharmaceutical Sciences, School of Pharmacy , University of Maryland , Baltimore , Maryland 20742 , United States.

Infectious diseases have a substantial global health impact. Clinicians need rapid and accurate diagnoses of infections to direct patient treatment and improve antibiotic stewardship. Current technologies employed in routine diagnostics are based on bacterial culture followed by morphological trait differentiation and biochemical testing, which can be time-consuming and labor-intensive. With advances in mass spectrometry (MS) for clinical diagnostics, the U.S. Food and Drug Administration has approved two microbial identification platforms based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of microbial proteins. We recently reported a novel and complementary approach by comparing MALDI-TOF mass spectra of microbial membrane lipid fingerprints to identify ESKAPE pathogens. However, this lipid-based approach used a sample preparation method that required more than a working day from sample collection to identification. Here, we report a new method that extracts lipids efficiently and rapidly from microbial membranes using an aqueous sodium acetate (SA) buffer that can be used to identify clinically relevant Gram-positive and -negative pathogens and fungal species in less than an hour. The SA method also has the ability to differentiate antibiotic-susceptible and antibiotic-resistant strains, directly identify microbes from biological specimens, and detect multiple pathogens in a mixed sample. These results should have positive implications for the manner in which bacteria and fungi are identified in general hospital settings and intensive care units.
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http://dx.doi.org/10.1021/acs.analchem.8b02611DOI Listing
January 2019

A Prospective Study of Complex Isolates and Colistin Susceptibility Monitoring by Mass Spectrometry of Microbial Membrane Glycolipids.

J Clin Microbiol 2019 03 27;57(3). Epub 2019 Feb 27.

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland Baltimore, Baltimore, Maryland, USA

is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics.
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http://dx.doi.org/10.1128/JCM.01100-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425172PMC
March 2019

Pathogen Identification Direct From Polymicrobial Specimens Using Membrane Glycolipids.

Sci Rep 2018 10 26;8(1):15857. Epub 2018 Oct 26.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, 21201, USA.

With the increased prevalence of multidrug-resistant Gram-negative bacteria, the use of colistin and other last-line antimicrobials is being revisited clinically. As a result, there has been an emergence of colistin-resistant bacterial species, including Acinetobacter baumannii and Klebsiella pneumoniae. The rapid identification of such pathogens is vitally important for the effective treatment of patients. We previously demonstrated that mass spectrometry of bacterial glycolipids has the capacity to identify and detect colistin resistance in a variety of bacterial species. In this study, we present a machine learning paradigm that is capable of identifying A. baumannii, K. pneumoniae and their colistin-resistant forms using a manually curated dataset of lipid mass spectra from 48 additional Gram-positive and -negative organisms. We demonstrate that these classifiers detect A. baumannii and K. pneumoniae in isolate and polymicrobial specimens, establishing a framework to translate glycolipid mass spectra into pathogen identifications.
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http://dx.doi.org/10.1038/s41598-018-33681-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203844PMC
October 2018

Rapid Food Product Analysis by Surface Acoustic Wave Nebulization Coupled Mass Spectrometry.

Food Anal Methods 2018 Sep 21;11(9):2447-2454. Epub 2018 Mar 21.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA.

Rapid food product analysis is of great interest for quality control and assurance during the production process. Conventional quality control protocols require time and labor intensive sample preparation for analysis by state-of-the-art analytical methods. To reduce overall cost and facilitate rapid qualitative assessments, food products need to be tested with minimal sample preparation. We present a novel and simple method for assessing food product compositions by mass spectrometry using a novel surface acoustic wave nebulization method. This method provides significant advantages over conventional methods requiring no pumps, capillaries, or additional chemicals to enhance ionization for mass spectrometric analysis. In addition, the surface acoustic wave nebulization - mass spectrometry method is ideal for rapid analysis and to investigate certain compounds by using the mass spectra as a type of species-specific fingerprint analysis. We present for the first time surface acoustic wave nebulization generated mass spectra of a variety of fermented food products from a small selection of vinegars, wines, and beers.
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http://dx.doi.org/10.1007/s12161-018-1232-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157634PMC
September 2018

Mass Spectrometry-based Structural Analysis and Systems Immunoproteomics Strategies for Deciphering the Host Response to Endotoxin.

J Mol Biol 2018 08 24;430(17):2641-2660. Epub 2018 Jun 24.

Laboratory of Immune System Biology (LISB), National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD 20814, USA. Electronic address:

One cause of sepsis is systemic maladaptive immune response of the host to bacteria and specifically, to Gram-negative bacterial outer-membrane glycolipid lipopolysaccharide (LPS). On the host myeloid cell surface, proinflammatory LPS activates the innate immune system via Toll-like receptor-4/myeloid differentiation factor-2 complex. Intracellularly, LPS is also sensed by the noncanonical inflammasome through caspase-11 in mice and 4/5 in humans. The minimal functional determinant for innate immune activation is the membrane anchor of LPS called lipid A. Even subtle modifications to the lipid A scaffold can enable, diminish, or abolish immune activation. Bacteria are known to modify their LPS structure during environmental stress and infection of hosts to alter cellular immune phenotypes. In this review, we describe how mass spectrometry-based structural analysis of endotoxin helped uncover major determinations of molecular pathogenesis. Through characterization of LPS modifications, we now better understand resistance to antibiotics and cationic antimicrobial peptides, as well as how the environment impacts overall endotoxin structure. In addition, mass spectrometry-based systems immunoproteomics approaches can assist in elucidating the immune response against LPS. Many regulatory proteins have been characterized through proteomics and global/targeted analysis of protein modifications, enabling the discovery and characterization of novel endotoxin-mediated protein translational modifications.
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http://dx.doi.org/10.1016/j.jmb.2018.06.032DOI Listing
August 2018

Serum Proteomic Profiling to Identify Biomarkers of Premature Carotid Atherosclerosis.

Sci Rep 2018 06 15;8(1):9209. Epub 2018 Jun 15.

Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Turku, Finland.

To evaluate the presence of serum protein biomarkers associated with the early phases of formation of carotid atherosclerotic plaques, label-free quantitative proteomics analyses were made for serum samples collected as part of The Cardiovascular Risk in Young Finns Study. Samples from subjects who had an asymptomatic carotid artery plaque detected by ultrasound examination (N = 43, Age = 30-45 years) were compared with plaque free controls (N = 43) (matched for age, sex, body weight and systolic blood pressure). Seven proteins (p < 0.05) that have been previously linked with atherosclerotic phenotypes were differentially abundant. Fibulin 1 proteoform C (FBLN1C), Beta-ala-his-dipeptidase (CNDP1), Cadherin-13 (CDH13), Gelsolin (GSN) and 72 kDa type IV collagenase (MMP2) were less abundant in cases, whereas Apolipoproteins C-III (APOC3) and apolipoprotein E (APOE) were more abundant. Using machine learning analysis, a biomarker panel of FBLN1C, APOE and CDH13 was identified, which classified cases from controls with an area under receiver-operating characteristic curve (AUROC) value of 0.79. Furthermore, using selected reaction monitoring mass spectrometry (SRM-MS) the decreased abundance of FBLN1C was verified. In relation to previous associations of FBLN1C with atherosclerotic lesions, the observation could reflect its involvement in the initiation of the plaque formation, or represent a particular risk phenotype.
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http://dx.doi.org/10.1038/s41598-018-27265-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003912PMC
June 2018

Dataset describing the development, optimization and application of SRM/MRM based targeted proteomics strategy for quantification of potential biomarkers of EGFR TKI sensitivity.

Data Brief 2018 Aug 2;19:424-436. Epub 2018 May 2.

Thoracic & Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, Bethesda, MD, United States.

The data presented here describes the use of targeted proteomic assays to quantify potential biomarkers of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) sensitivity in lung adenocarcinoma and is related to the research article: "Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma" [1]. This article describes the data associated with liquid chromatography coupled to multiple reaction monitoring (LC-MRM) method development which includes selection of an optimal transition list, retention time prediction and building of reverse calibration curves. Sample preparation and optimization which includes phosphotyrosine peptide enrichment via a combination of pan-phosphotyrosine antibodies is described. The dataset also consists of figures, tables and Excel files describing the quantitative results of testing these optimized methods in two lung adenocarcinoma cell lines with EGFR mutations.
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http://dx.doi.org/10.1016/j.dib.2018.04.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997585PMC
August 2018

Quantitative proteomic characterization and comparison of T helper 17 and induced regulatory T cells.

PLoS Biol 2018 05 31;16(5):e2004194. Epub 2018 May 31.

Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.

The transcriptional network and protein regulators that govern T helper 17 (Th17) cell differentiation have been studied extensively using advanced genomic approaches. For a better understanding of these biological processes, we have moved a step forward, from gene- to protein-level characterization of Th17 cells. Mass spectrometry-based label-free quantitative (LFQ) proteomics analysis were made of in vitro differentiated murine Th17 and induced regulatory T (iTreg) cells. More than 4,000 proteins, covering almost all subcellular compartments, were detected. Quantitative comparison of the protein expression profiles resulted in the identification of proteins specifically expressed in the Th17 and iTreg cells. Importantly, our combined analysis of proteome and gene expression data revealed protein expression changes that were not associated with changes at the transcriptional level. Our dataset provides a valuable resource, with new insights into the proteomic characteristics of Th17 and iTreg cells, which may prove useful in developing treatment of autoimmune diseases and developing tumor immunotherapy.
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http://dx.doi.org/10.1371/journal.pbio.2004194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979006PMC
May 2018

Correction to: Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

J Am Soc Mass Spectrom 2018 06;29(6):1230

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Pharmacy Hall North Room 623, 20 N. Pine St, Baltimore, MD, 21201, USA.

In the preceding article "Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate" by Oyler et al., an error in the J5 E. coli LPS chemical structure (Figs. 2 and 4) was introduced and propagated into the final revision.
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http://dx.doi.org/10.1007/s13361-018-1960-8DOI Listing
June 2018

Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma.

J Proteomics 2018 10 13;189:48-59. Epub 2018 Apr 13.

Thoracic & Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, Bethesda, MD, United States. Electronic address:

Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30-40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFR mutation. However, in H1975 cells, which harbor the TKI-resistant EGFR mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. SIGNIFICANCE: In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and correlated the results of the relative quantification with amounts estimated from the calibration curves. This approach represents a way to validate and verify biomarker candidates discovered from large-scale global phospho-proteomics analysis. The application of these modified immuno-MRM assays in lung adenocarcinoma cells provides proof-of concept for the feasibility of clinical applications. These assays may be used in prospective clinical studies of EGFR TKI treatment of EGFR mutant lung cancer to correlate treatment response and other clinical endpoints.
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http://dx.doi.org/10.1016/j.jprot.2018.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984720PMC
October 2018

Host-pathogen dynamics through targeted secretome analysis of stimulated macrophages.

J Proteomics 2018 10 20;189:34-38. Epub 2018 Mar 20.

Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

The pattern recognition receptors (PRRs) facilitate an organism's first line of defense against interlopers and shape the overall innate immune response through sensing and sampling pathogen-associated molecular patterns (PAMPs). The Toll-like receptor (TLR) family is the prototypic PRR family. Upon recognition of PAMPs, TLRs promote MyD88 dependent and independent responses. Understanding how different PAMPs are recognized by their specific TLRs and how pathogen recognition initiates immune activation is an intense area of research. Previously, we have reported the discovery of the temporal changes in signaling cascades of macrophage proteome and secretome post-stimulation with three different PAMPs. To extend our global proteomics approach to targeted protein abundance quantification, we describe the macrophage secretome targeted proteomics assay. We chose three different pathogens that specifically stimulate diverse TLRs (TLR2, TLR4, and TLR7). Using a simple targeted proteomics approach, combining data-dependent acquisition with an inclusion list, an array of cytokines, chemokines, and transcription factors can be profiled for their secretome abundance. This strategy facilitates the profiling and validation of pathogen-specific temporal changes in the macrophage secretome.
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http://dx.doi.org/10.1016/j.jprot.2018.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149218PMC
October 2018