Proteomics in Forensic Analysis: Applications for Human Samples
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Standard Flow Multiplexed Proteomics (Sflompro) – an Accessible and Cost-Effective Alternative to Nanolc Workflows
bioRxiv preprint doi: https://doi.org/10.1101/2020.02.25.964379; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Standard Flow Multiplexed Proteomics (SFloMPro) – An Accessible and Cost-Effective Alternative to NanoLC Workflows Conor Jenkins1 and Ben Orsburn2* 1Hood College Department of Biology, Frederick, MD 2University of Virginia Medical School, Charlottesville, VA *To whom correspondence should be addressed; [email protected] Abstract Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. -
W Yang Et Al. Proteomic Approaches to the Analysis of Multiprotein
832 DOI 10.1002/pmic.200700650 Proteomics 2008, 8, 832–851 REVIEW Proteomic approaches to the analysis of multiprotein signaling complexes Wei Yang1, 2, Hanno Steen3 and Michael R. Freeman1, 2 1 The Urological Diseases Research Center, Department of Urology, Children’s Hospital Boston, Boston, MA, USA 2 Departments of Surgery, Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA 3 Department of Pathology, Harvard Medical School and Children’s Hospital Boston, Boston, MA, USA Signal transduction is one of the most active fields in modern biomedical research. Increasing Received: July 9, 2007 evidence has shown that signaling proteins associate with each other in characteristic ways to Revised: October 23, 2007 form large signaling complexes. These diverse structures operate to boost signaling efficiency, Accepted: October 23, 2007 ensure specificity and increase sensitivity of the biochemical circuitry. Traditional methods of protein analysis are inadequate to fully characterize and understand these structures, which are intricate, contain many components and are highly dynamic. Instead, proteomics technologies are currently being applied to investigate the nature and composition of multimeric signaling complexes. This review presents commonly used and potential proteomic methods of analyzing diverse protein complexes along with a discussion and a brief evaluation of alternative ap- proaches. Challenges associated with proteomic analysis of signaling complexes are also dis- cussed. Keywords: Cross-linking / Mass spectrometry / Post-translational modification / Quantitative proteomics / Signaling complexes 1 Introduction 1980s, astonishingly rapid progress has been made in understanding the mechanisms of signal transduction. The modern field of cell signaling can be traced back to the Many old concepts have been abandoned or revised and mid-1950s, when it was discovered that reversible phospho- new ones have emerged. -
ITRAQ-Based Quantitative Proteomic Analysis of Processed Euphorbia Lathyris L
Zhang et al. Proteome Science (2018) 16:8 https://doi.org/10.1186/s12953-018-0136-6 RESEARCH Open Access ITRAQ-based quantitative proteomic analysis of processed Euphorbia lathyris L. for reducing the intestinal toxicity Yu Zhang1, Yingzi Wang1*, Shaojing Li2*, Xiuting Zhang1, Wenhua Li1, Shengxiu Luo1, Zhenyang Sun1 and Ruijie Nie1 Abstract Background: Euphorbia lathyris L., a Traditional Chinese medicine (TCM), is commonly used for the treatment of hydropsy, ascites, constipation, amenorrhea, and scabies. Semen Euphorbiae Pulveratum, which is another type of Euphorbia lathyris that is commonly used in TCM practice and is obtained by removing the oil from the seed that is called paozhi, has been known to ease diarrhea. Whereas, the mechanisms of reducing intestinal toxicity have not been clearly investigated yet. Methods: In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method was applied to investigate the effects of Euphorbia lathyris L. on the protein expression involved in intestinal metabolism, in order to illustrate the potential attenuated mechanism of Euphorbia lathyris L. processing. Differentially expressed proteins (DEPs) in the intestine after treated with Semen Euphorbiae (SE), Semen Euphorbiae Pulveratum (SEP) and Euphorbiae Factor 1 (EFL1) were identified. The bioinformatics analysis including GO analysis, pathway analysis, and network analysis were done to analyze the key metabolic pathways underlying the attenuation mechanism through protein network in diarrhea. Western blot were performed to validate selected protein and the related pathways. Results: A number of differentially expressed proteins that may be associated with intestinal inflammation were identified. -
Keratins Couple with the Nuclear Lamina and Regulate Proliferation in Colonic Epithelial Cells Carl-Gustaf A
bioRxiv preprint doi: https://doi.org/10.1101/2020.06.22.164467; this version posted June 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Keratins couple with the nuclear lamina and regulate proliferation in colonic epithelial cells Carl-Gustaf A. Stenvall1*, Joel H. Nyström1*, Ciarán Butler-Hallissey1,5, Stephen A. Adam2, Roland Foisner3, Karen M. Ridge2, Robert D. Goldman2, Diana M. Toivola1,4 1 Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland 2 Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA 3 Max Perutz Labs, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria 4 Turku Center for Disease Modeling, Turku, Finland 5 Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland * indicates equal contribution Running Head: Colonocyte keratins couple to nuclear lamina Corresponding author: Diana M. Toivola Cell Biology/Biosciences, Faculty of Science and Engineering, Åbo Akademi University Tykistökatu 6A, FIN-20520 Turku, Finland Telephone: +358 2 2154092 E-mail: [email protected] Keywords: Keratins, lamin, intermediate filament, colon epithelial cells, LINC proteins, proliferation, pRb, YAP bioRxiv preprint doi: https://doi.org/10.1101/2020.06.22.164467; this version posted June 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. -
Keratin 1 Maintains Skin Integrity and Participates in an Inflammatory
Research Article 5269 Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18 Wera Roth1, Vinod Kumar1, Hans-Dietmar Beer2, Miriam Richter1, Claudia Wohlenberg3, Ursula Reuter3, So¨ ren Thiering1, Andrea Staratschek-Jox4, Andrea Hofmann4, Fatima Kreusch4, Joachim L. Schultze4, Thomas Vogl5, Johannes Roth5, Julia Reichelt6, Ingrid Hausser7 and Thomas M. Magin1,* 1Translational Centre for Regenerative Medicine (TRM) and Institute of Biology, University of Leipzig, 04103 Leipzig, Germany 2University Hospital, Department of Dermatology, University of Zurich, 8006 Zurich, Switzerland 3Institute of Biochemistry and Molecular Biology, Division of Cell Biochemistry, University of Bonn, 53115 Bonn, Germany 4Department of Genomics and Immunoregulation, LIMES Institute, University of Bonn, 53115 Bonn, Germany 5Institute of Immunology, University of Mu¨nster, 48149 Mu¨nster, Germany 6Institute of Cellular Medicine and North East England Stem Cell Institute, Newcastle University, Newcastle upon Tyne NE2 4HH, UK 7Universita¨ts-Hautklinik, Ruprecht-Karls-Universita¨t Heidelberg, 69120 Heidelberg, Germany *Author for correspondence ([email protected]) Accepted 8 October 2012 Journal of Cell Science 125, 5269–5279 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.116574 Summary Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. KRT1 mutations cause epidermolytic ichthyosis in humans, characterized by loss of barrier integrity and recurrent erythema. In search of the largely unknown pathomechanisms and the role of keratins in barrier formation and inflammation control, we show here that Krt1 is crucial for maintenance of skin integrity and participates in an inflammatory network in murine keratinocytes. -
Impact of Keratin Network Regulation on Migrating Cells
Impact of Keratin Network Regulation on Migrating Cells Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Anne Pora, Ingénieur, Master aus Rueil-Malmaison, Frankreich Berichter: Univ.-Prof. Dr. Björn Kampa Univ.-Prof. Dr. med. Rudolf Leube Tag der mündlichen Prüfung: 02.04.19 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. This work was performed at the Institute for Molecular and Cellular Anatomy at University Hospital RWTH Aachen by the mentorship of Prof. Dr. med. Rudolf E. Leube. It was exclusively performed by myself, unless otherwise stated in the text. 1. Reviewer: Univ.-Prof. Dr. Björn Kampa 2. Reviewer: Univ.-Prof. Dr. med. Rudolf E. Leube Toulouse (FR), 30.11.18 2 Table of Contents Table of Contents 3 Chapter 1: Introduction 6 1. Cell migration 6 A key process in physiological and pathological conditions 6 Different kinds of migration 6 Influence of the environment 7 2. The cytoskeleton: a key player in cell migration 9 The cytoskeleton 9 Actin and focal adhesions 9 Microtubules 12 Keratin intermediate filaments and hemidesmosomes 13 Cross-talk between keratin intermediate filaments and others 22 cytoskeletal components Imaging cytoskeletal dynamics in migrating cells 24 3. Objectives 26 Chapter 2: Materials and Methods 27 1. Cell culture conditions 27 2. Keratin extraction and immunoblotting 28 3. Immunofluorescence 32 4. Plasmid constructs and DNA transfection into cultured cells 34 5. Drug treatments 35 6. Micropatterning 35 7. Preparation of elastic substrates 36 8. Imaging conditions 36 3 9. -
Biological Functions of Cytokeratin 18 in Cancer
Published OnlineFirst March 27, 2012; DOI: 10.1158/1541-7786.MCR-11-0222 Molecular Cancer Review Research Biological Functions of Cytokeratin 18 in Cancer Yu-Rong Weng1,2, Yun Cui1,2, and Jing-Yuan Fang1,2,3 Abstract The structural proteins cytokeratin 18 (CK18) and its coexpressed complementary partner CK8 are expressed in a variety of adult epithelial organs and may play a role in carcinogenesis. In this study, we focused on the biological functions of CK18, which is thought to modulate intracellular signaling and operates in conjunction with various related proteins. CK18 may affect carcinogenesis through several signaling pathways, including the phosphoinosi- tide 3-kinase (PI3K)/Akt, Wnt, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathways. CK18 acts as an identical target of Akt in the PI3K/Akt pathway and of ERK1/2 in the ERK MAPK pathway, and regulation of CK18 by Wnt is involved in Akt activation. Finally, we discuss the importance of gaining a more complete understanding of the expression of CK18 during carcinogenesis, and suggest potential clinical applications of that understanding. Mol Cancer Res; 10(4); 1–9. Ó2012 AACR. Introduction epithelial organs, such as the liver, lung, kidney, pancreas, The intermediate filaments consist of a large number of gastrointestinal tract, and mammary gland, and are also nuclear and cytoplasmic proteins that are expressed in a expressed by cancers that arise from these tissues (7). In the tissue- and differentiation-dependent manner. The compo- absence of CK8, the CK18 protein is degraded and keratin fi intermediate filaments are not formed (8). -
Challenges in Clinical Metaproteomics Highlighted by the Analysis of Acute Leukemia Patients with Gut Colonization by Multidrug-Resistant Enterobacteriaceae
proteomes Article Challenges in Clinical Metaproteomics Highlighted by the Analysis of Acute Leukemia Patients with Gut Colonization by Multidrug-Resistant Enterobacteriaceae Julia Rechenberger 1,† , Patroklos Samaras 1,†, Anna Jarzab 1, Juergen Behr 2, Martin Frejno 1 , Ana Djukovic 3, Jaime Sanz 4,5, Eva M. González-Barberá 4, Miguel Salavert 4, Jose Luis López-Hontangas 4, Karina B. Xavier 6 , Laurent Debrauwer 7,8, Jean-Marc Rolain 9, Miguel Sanz 4,5 , Marc Garcia-Garcera 10, Mathias Wilhelm 1,* , Carles Ubeda 3,11,* and Bernhard Kuster 1,2,* 1 Chair of Proteomics and Bioanalytics, Technical University of Munich, 85354 Freising, Germany; [email protected] (J.R.); [email protected] (P.S.); [email protected] (A.J.); [email protected] (M.F.) 2 Bavarian Center for Biomolecular Mass Spectrometry, Technical University of Munich, 85354 Freising, Germany; [email protected] 3 Centro Superior de Investigación en Salud Pública-FISABIO, 46020 Valencia, Spain; [email protected] 4 Hospital Universitari i Politècnic La Fe, 46026 Valencia, Spain; [email protected] (J.S.); [email protected] (E.M.G.-B.); [email protected] (M.S.); [email protected] (J.L.L.-H.); [email protected] (M.S.) 5 CIBERONC, Instituto Carlos III, 28029 Madrid, Spain 6 Instituto Gulbenkian de Ciência, 2780 Oeiras, Portugal; [email protected] 7 Toxalim, Université de Toulouse, INRA, INP-ENVT, INP-EI-Purpan, Université de Toulouse 3 Paul Sabatier, 31027 Toulouse, France; [email protected] 8 Axiom Platform, UMR 1331 Toxalim, MetaToul-MetaboHUB, -
Cytokeratins 6 and 16 Are Frequently Expressed in Head and Neck Squamous Cell Carcinoma Cell Lines and Fresh Biopsies
ANTICANCER RESEARCH 25: 2675-2680 (2005) Cytokeratins 6 and 16 are Frequently Expressed in Head and Neck Squamous Cell Carcinoma Cell Lines and Fresh Biopsies ANDREAS M. SESTERHENN, ROBERT MANDIC, ANJA A. DÜNNE and JOCHEN A. WERNER Department of Otolaryngology, Head and Neck Surgery, Philipps-University of Marburg, Deutschhausstrasse 3, 35037 Marburg, Germany Abstract. Background: Cytokeratins (CK) are members of Together with actin-microfilaments and microtubules, keratin intermediate filaments, which are predominantly found in filaments form the cytoskeletons of vertebrate epithelial cells epithelial cells. Different types of epithelia are characterized by (1). Cytokeratins are subclassified into 20 different a distinct composition of CK. Recently immunohistochemical polypeptides (CKs 1-20) (3). CK’s are expressed in various investigations demonstrated that, among others, CKs 6, 14, 16 combinations depending on the epithelial cell type (3-5). They and 17 are regularly expressed in benign stratified squamous are subdivided into two sequence types (I and II) that are epithelium of the head and neck as well as in squamous cell typically, but not necessarily, coexpressed as specific pairs with carcinoma of the head and neck (HNSCC) in contrast to CKs complex expression patterns (1, 5). Except for CK18, all genes 1, 10 and 11, that were only rarely expressed in these tissues. of type-I-CKs are localized on chromosome 17, whereas the Materials and Methods: Total RNA was isolated from 15 genes from type-II-CKs are localized on chromosome 12 (3). primary cell lines derived from HNSCC and from 15 tissue CKs were found to be specific in distinct constellation for samples of oro- and hypopharyngeal carcinomas obtained different types of epithelia (5). -
Terrestrial Vertebrates Have Two Keratin Gene Clusters; Striking Differences in Teleost fish Alexander Zimek, Klaus Weberã
ARTICLE IN PRESS European Journal of Cell Biology 84 (2005) 623–635 www.elsevier.de/ejcb Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish Alexander Zimek, Klaus Weberà Department of Biochemistry and Cell Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Go¨ttingen, Germany Received 16 December 2004; received in revised form 25 January 2005; accepted 25 January 2005 Abstract Keratins I and II form the largest subgroups of mammalian intermediate filament (IF) proteins and account as obligatory heteropolymers for the keratin filaments of epithelia. All human type I genes except for the K18 gene are clustered on chromosome 17q21, while all type II genes form a cluster on chromosome 12q13, that ends with the type I gene K18. Highly related keratin gene clusters are found in rat and mouse. Since fish seem to lack a keratin II cluster we screened the recently established draft genomes of a bird (chicken) and an amphibian (Xenopus). The results show that keratin I and II gene clusters are a feature of all terrestrial vertebrates. Because hair with its multiple hair keratins and inner root sheath keratins is a mammalian acquisition, the keratin gene clusters of chicken and Xenopus tropicalis have only about half the number of genes found in mammals. Within the type I clusters all genes have the same orientation. In type II clusters there is a rare gene of opposite orientation. Finally we show that the genes for keratins 8 and 18, which are the first expression pair in embryology, are not only adjacent in mammals, but also in Xenopus and three different fish. -
Identification of Keratins and Analysis of Their Expression in Carp and Goldfish: Comparison with the Zebrafish and Trout Keratin Catalog
Cell Tissue Res DOI 10.1007/s00441-005-0031-1 REGULAR ARTICLE Dana M. García . Hermann Bauer . Thomas Dietz . Thomas Schubert . Jürgen Markl . Michael Schaffeld Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog Received: 21 March 2005 / Accepted: 23 May 2005 # Springer-Verlag Abstract With more than 50 genes in human, keratins zebrafish, and the cyprinid fishes being more similar to each make up a large gene family, but the evolutionary pres- other than to the salmonid trout. Because of the detected sure leading to their diversity remains largely unclear. Nev- similarity of keratin expression among the cyprinid fishes, ertheless, this diversity offers a means to examine the we propose that, for certain experiments, they are inter- evolutionary relationships among organisms that express changeable. Although the zebrafish distinguishes itself as keratins. Here, we report the analysis of keratins expressed being a developmental and genetic/genomic model organ- in two cyprinid fishes, goldfish and carp, by two-dimen- ism, we have found that the goldfish, in particular, is a more sional polyacrylamide gel electrophoresis, complementary suitable model for both biochemical and histological stud- keratin blot binding assay, and immunoblotting. We further ies of the cytoskeleton, especially since goldfish cytoskel- explore the expression of keratins by immunofluorescence etal preparations seem to be more resistant to degradation microscopy. Comparison is made with the keratin expres- than those from carp or zebrafish. sion and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of mo- Keywords Keratin . -
Types I and II Keratin Intermediate Filaments
Downloaded from http://cshperspectives.cshlp.org/ on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press Types I and II Keratin Intermediate Filaments Justin T. Jacob,1 Pierre A. Coulombe,1,2 Raymond Kwan,3 and M. Bishr Omary3,4 1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 2Departments of Biological Chemistry, Dermatology, and Oncology, School of Medicine, and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland 21205 3Departments of Molecular & Integrative Physiologyand Medicine, Universityof Michigan, Ann Arbor, Michigan 48109 4VA Ann Arbor Health Care System, Ann Arbor, Michigan 48105 Correspondence: [email protected] SUMMARY Keratins—types I and II—are the intermediate-filament-forming proteins expressed in epithe- lial cells. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type–, differentiation-, and context-dependent manner. Here, we review how keratins serve multiple homeostatic and stress-triggered mechanical and nonmechanical functions, including maintenance of cellular integrity, regulation of cell growth and migration, and protection from apoptosis. These functions are tightly regulated by posttranslational modifications and keratin-associated proteins. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility or altered