1 PROTEOMIC SAMPLE PREPARATION Two-dimensional electrophoresis Hana Konečná Proteomics Core Facility CEITEC Central European Institute of Technology NCBR National Centre for Biomolecular Research protein sample complex peptide mixture complex protein sample simple peptide mixture simple database identification quantification electrophoresis centrifugation chromatography MS MS/MS modification characterization shotgun bottom-up I. SEPARATION II. PREFRACTIONATION Two-dimensonal electrophoresis 2-DE ? Proteomický experiment extrakce fokusace SDS-PAGE digest MS identifikace neznámý protein 2DE 1DE Proteomic experiment extraction focusing SDS-PAGE digest MS identification unknown protein HOMOGENIZATION • mechanical • ultrasound • pressure • freeze/thaw lysis • detergent lysis Název prezentace v zápatí 6 CryoMill Liquid Nitrogene Watch for keratins! SAMPLE PREPARATION Solubilization urea thiourea detergents Reduction DTT TBP TCEP dithiotreitol tributylphospine Tris (2-carboxyethyl) phosphine hydrochloride Inhibition of proteases, phosphatases glycosylases Contaminants removal Protein assay DETERGENTS  no net charge  0.5 – 4%  working in high urea  non ionogenic  zwitterion  SDS only up to 0.25% RULE OF THUMB  avoid proteolysis  simple preparation  fresh reagents  fresh sample  remove particles - spin  remove contaminants CONTAMINANTS  salts, buffers  small molecules  ionic detergents  nucleic acids  polysaccharides  lipids  phenols 2-DE • first dimension • second dimension IEF SDS-PAGE 1. ROZMĚR IZOELEKTRICKÁ FOKUSACE migrace nabitých částic v gradientu pH v elektrickém poli 1st dimension ISOELECTRIC FOCUSING migration of charged molecules in pH gradient in electric field ISOELECTRIC FOCUSING • immobilized pH gradient • ampholytes + + + - - - ! RANGE OF STRIP SIZE OF STRIP RANGE OF STRIP denaturace SDS  redukce DTT  alkylace IAA  EKVILIBRACE STRIPU !EQUILIBRATION OF STRIP denaturation reduction alkylation 2. ROZMĚR SDS-PAGE migrace aniontů v elektrickém poli podle MW !2nd dimension SDS-PAGE Migration of anions in electric field according to MW STRIP GEL + - + FOKUSACE SDS-PAGE - equilibration Gel orientation ACIDIC pI BASIC HIGH MW LOW !FOCUSING 2-DE INSTRUMENTATION  Protean IEF  Protean Dodeca Cell  Densitometer GS-800  FLA-7000, STORM PDQuest, Quantity One Protean Plus Dodeca Cell Mini-Protean 3 Dodeca Cell Protean II xi Cell PROTEIN DETECTION • gel x blot • visualisation staining radioactivity assay immunodetection • staining in gel  post-electrophoretic  pre-electrophoretic  protein specific  PTM specific  visible spectrum  fluorescence Sypro Ruby Coomassie silver 1.4 ng 36 ng 0.6 ng PROTEIN DETECTION IN GEL PTM specific staining Pro-Q Diamond Pro-Q Emerald PROTEIN STAINING – LINEARITY Sypro Ruby Ag Sypro Ruby • quality • quantity IMAGE ANALYSIS biological replicates technical replicates biological variability technical variability three plantelets analysed same plantelet analysed separately under same conditions three times under same conditions 2D or not 2D ?  visual aspects  reproducibility  dynamic range  extreme proteins (membrane, basic...)  difficult automatization  postdigestion extraction MULTIDIMENSIONAL CHROMATOGRAPHY  large sample volumes  on-column concentration  membrane proteins, basic proteins  no staining  peptides – going directly to MS  automatization  vizual aspects lost: pI a Mr  LC - serial analysis  GE - more samples in paralel AGAINST FOR Difference Gel Electrophoresis DIGE BIOMARKERS … NEEDLE IN HAYSTACKS prefractionation ▪ separation ▪ identification ▪ control vs. sample • haystack - proteins without relation to disease • neeedles – disease specific proteins • potencial needles difficult to validate biological variability! • are needles worth further examination? • often contain PTM, difficult to be identified by MS Day 21 – before clinical manifestation Day 44 – after clinical manifestation Biomarkery v lidské plasmě separation identification Biomarkers in human plasma DIGEST • IN-GEL • IN-SOLUTIONtrypsin Glu-C Asp-N thermolysin MAVEPFPRRPITRPHASIEVDTSGTGGSAGSSEKVF CLIGQAEGGEPNTVYELRNYAQAKRLFRSGELLDAI ELAWGSNPNYTAGRILAMRIEDAKPASAEIGGLKIT SKIYGNVANNIQVGLEKNTLSDSLRLRVIFQDDRFN EVYDNIGNIFTIKYKGEEANATFSVEHDEETQKASR LVLKVGDQEVKSYDLTGGAYDYTNAIITDINQLPDF EAKLSPFGDKNLESSKLDKIENANIKDKAVYVKAVF GDLEKQTAYNGIVSFEQLNAEGEVPSNVEVEAGEE SATVTATSPIKTIEPFELTKLKGGTNGEPPATWADKL DKFAHEGGYYIVPLSSKQSVHAEVASFVKERSDAGE PMRAIVGGGFNESKEQLFGRQASLSNPRVSLVANS GTFVMDDGRKNHVPAYMVAVALGGLASGLEIGES ITFKPLRVSSLDQIYESIDLDELNENGIISIEFVRNRTN TFFRIVDDVTTFNDKSDPVKAEMAVGEANDFLVSE LKVQLEDQFIGTRTINTSASIIKDFIQSYLGRKKRDN EIQDFPAEDVQVIVEGNEARISMTVYPIRSFKKISVS LVYKQQTLQA MS G I G O GARBAGE IN - GARBAGE OUT G I G O • R.M. Twyman: Principles of Proteomics • R.Westermeier, T.Naven, H-R Höpker: Proteomics in Practice • A.J.Link: 2D Proteome Analysis Protocols • Current Protocols in Protein Science • R.J.Simpson: Proteins and Proteomics • T.Rabilloud: Proteome Research: Two-dimensional Gel • Electrophoresis and Identification Methods • A. Görg, W. Weiss, M.J.Dunn: Proteomics 2004, 4, 3665, rev. • I. Miller, J. Crawford, E. Gianazza: Proteomics 2006, 6, rev. • F.Chevalier: Proteome Science 2010, 8:23, review • R. Burgess, M. Deutscher: Guide to Protein Purification LITERATURE I. SEPARATION II. PREFRACTIONATION GENOME PROTEOME ISOFORMS PTM ~200 variants (fosforylation, glykosylation, acylation, methylation…) CONCENTRATION RANGE ~ 10 orders of magnitude PREFRACTIONATION MS 10 000km 100km 10km 10cm 1 000km 1km 100m 10m 1m 1cm Abundant proteins in human plasma Removal of abundant proteins by affinity chromatography HSA IgG AFFINITY DEPLETION Apolipoprotein Immunoglobulin kappa light chain Immunoglobulin light chain albumin Lidská plazma - vázaná frakce po afinitní depleci ALBUMIN IgG Barvení: CB G-250 Human plasma – bound fractions after affinity depletion Staining CBB G-250 CPPL Combinatorial Peptide Ligand Library PROTEOMINERPREFRACTIONATION PREFRAKCIONACE MicroRotofor OffGel Fractionator IEF • prefractionation in solution • prefractionation in solution using IPG strip prefractionation offgel Beer: 70 proteins OFFGEL IEF prefractionation of proteins or peptides Protein digest IEF of mixed proteins on the strip fractions of strip IPG-IEF Blue Native Electrophoresis BNE • Separation of native proteins • Separation of membrane complexes • Solubilization by non-ionic detergents • Charged by Coomassie G-250 • BN PAGE gel (strip/band) as 2D G-250 SDS 2DE BNE SDS-PAGE PREFRAKCIONACE MIKRO ROZSAHY pIPREFRACTIONATION MICRO RANGES GEL CHROMATOGRAPHY SDS 8M Urea Peptides DTT IAA spin spin spin incubation spin proteins nucleic acids Lysate 8M Urea Ammonium Trypsin SDS DTT IAA bicarbonate PROTEINS PEPTIDES Nature Methods 6, 359 - 362 (2009) Filter Aided Sample Preparation FASPFASP Filter aided sample preparation 54 single-pot solid-phase-enhanced sample preparationSP3 “single vessel” approach: - surface-functionalized (i.e. carboxylate-coated) paramagnetic beads trap proteins and peptides in hydrophilic layers when the organic composition of the buffer is increased and the pH adjusted. - the beads can be immobilized within a magnetic field - efficient removal of contaminating agents including chaotropes and detergents by washingwith different organic solvents (i.e., ethanol and acetonitrile - after rinsing, bound proteins or peptides can be eluted from the beads using an aqueous solution. - protein cleanup, enzymatic digestion, desalting, and peptide recovery in a single tube. 55 iST in-StageTip method - complete sample preparation in a single reactor - resembles an in-solution digestion with the advantages of a single FASP-like reaction vessel that avoids the use of a filter membrane. - the C18 disk serves as a physical barrier for insoluble material and macromolecules. - additionally, it enables final peptide cleanup using solid-phase extraction (SPE). - One drawback of iST as compared to FASP is the limitation regarding the use of certain reagents (i.e., iST cannot remove SDS). “single vessel” approach MOTIVATING LITERATURE FOR ADVANCED READERS Two-dimensional gel electrophoresis in proteomics: A tutorial Thierry Rabilloud et al. Journal of Proteomics 2011 Two-dimensional gel electrophoresis in proteomics: past, present and future Thierry Rabilloud et al. Journal of Proteomics 2010 Proteomic biomarker discovery: It's more than just mass spectrometry Josip Blonder et al. Electrophoresis 2011 Basics and recent advances of two dimensional – polyacrylamide gel electrophoresis Sameh Magdeldin et al. Clinical Proteomics 2014 Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range Malte Sielaff et al. J. Proteome Res. 2017 For all the complex problems and difficult questions there is always one simple, easily comprehensible w r o n g answer.