Protein characterization by mass spectrometry C7250 Part V Zbyněk Zdráhal RG Proteomics, CEITEC-MU Proteomics CF, CEITEC-MU NCBR FS MU zdrahal@sci.muni.cz Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University mu1 nfsnf Proteomic MS applications File:Metabolomics schema.png http://en.wikipedia.org/wiki/File:Metabolomics_schema.png C7250 DNA protein functional protein mRNA posttranslational mods Protein complexes transcription posttranscriptional modifications (alternative splicing etc.) translation Proteomics – discipline dealing with proteome analysis what might happen what is really happening C7250 Proteomics - Why? * several proteins/proteoforms might form from each gene, not possible to indicate them by DNA/RNA analysis * there no direct correlation between mRNA content and final content of proteins * * functionality of protein depends frequently on its interaction with other proteins or DNA/RNA * * only at protein level epigenetics factors of gene expression regulation are detectable * C7250 self-portrait-or-desperate-man-gustave-courbet The Desperate Man, Gustave Courbet, 1844-45 Proteome analysis protein-X protein protein W-protein-Z X DNA-structure-and-bases characterization of all proteins including all their forms in cell (tissue, organisms) at given time under given conditions Genome C7250 Genome versus Proteome relatively stable all proteins including DNA sequence all their forms in cell (tissue, organisms) at given time under given conditions 4 basic building units 20 basic building units efficient analytical techniques necessity of development of developed (PCR, NGS) sensitive and reliable techniques for identification and quantitation C7250 Arabidopsis thaliana Mouse-ear cress arabi_bw1tr Genome 0.135 x 109 bp, ~ 27 000 geness 32 000 proteins C7250 http://www.uniprot.org/taxonomy/complete-proteomes human genome - 3.3 x 109 bp, ~ 21 000 genes http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GenomeSizes.html 54_evoluce „Evolution is strictly prohibited in this district“ Identification of microorganisms by MALDI-MS cultivation peptide/protein extraction MALDI MS (3 – 20 kDa) PCA analysis database of MALDI-MS profiles BD18215_ microorganism identification taxonomy MALDI-MS profiling C7250 5000 7000 9000 11000 m/z 0 500 1000 1500 2000 2500 a.i. 5000 7000 9000 11000 m/z 0 500 1000 1500 2000 2500 a.i. Alcaligenes faecalis Sphingomonas paucimobilis Aeromonas hydrophila Pseudomonas aeruginosa MALDI-MS spectra (profiles) of selected bacteria C7250 0 100 200 300 400 500 600 700 800 900 1000 Campylobacter fetus subsp fetus CCM 5683 CCM Campylobacter fetus subsp fetus CCM 6210 CCM Campylobacter fetus subsp fetus CCM 5682 CCM Campylobacter coli CCM 6211 CCM Campylobacter jejuni ssp jejuni CCM 6189 CCM Campylobacter jejuni ssp jejuni CCM 6191 CCM Campylobacter jejuni CCM 6214 CCM Campylobacter jejuni ssp jejuni CCM 7212 CCM Corynebacterium pilosum CCM 6140 CCM Corynebacterium urealyticum CCM 3975 CCM Corynebacterium urealyticum CCM 3976 CCM Corynebacterium urealyticum CCM 4186 CCM Finegoldia magna CCM 3785 CCM Streptococcus mitis CCM 7411 CCM Serratia rubidaea CCM 3412 CCM Peptostreptococcus anaerobius CCM 3790 CCM Staphylococcus epidermidis CCM 2124 CCM Staphylococcus epidermidis CCM 2446 CCM Staphylococcus epidermidis CCM 7221 CCM Staphylococcus saprophyticus CCM 3317 CCM Distance Level Graphical expression of MALDI-MS bacteria profile similarity L. Tvrzová, A. Teshim, I. Sedláček, M. Lexa, A. Voráč, O. Šedo,, A. Kostrzewa, T. Meier identification based on comparison of measured profile with database profile * validated method in clinical practise C7250 100 200 300 400 500 600 700 800 900 1000 Brewery 1 bottle 3 Brewery 1 bottle 4 Brewery 1 bottle 5 Brewery 1 bottle 1 Brewery 1 bottle 2 Brewery 2 bottle 3 Brewery 2 bottle 4 Brewery 2 bottle 5 Brewery 2 bottle 1 Brewery 2 bottle 2 Brewery 3 bottle 1 Brewery 3 bottle 2 Brewery 3 bottle 3 Brewery 3 bottle 4 Brewery 3 bottle 5 Distance Level MALDI-MS profiling of beer MALDI-TOF MS fingerprint containing proteins 0.5 1.0 1.5 2.0 2.5 4 x10 2000 3000 4000 5000 6000 7000 8000 9000 10000 cooperation with FCH BUT Brno prof. Márová C7250 14 cooperation with prof. Pekar, FS MU MALDI-MS profiling of spider venoms •evolution of food specialisation in spiders •species adaptations •ant-eating spiders Pekár S. et al., J. Anim. Ecol., 81 (4), 838-848 (2012) Bočánek O. et al., Toxicon, 133, 18-25 (2017) Pekár S. et al. Mol. Ecol., 27 (4), 1053-1064 (2018) Z. merlijni C7250 MALDI-MS profiling–early detection of diseases (peptide profiling, pattern profiling) BD18215_ MALDI MS, SELDI MS (3 – 20 kDa) profile analysis no or minimal sample prep (ionex, IMAC, affinity sorbents) * high number of factors influencing protein composition not related with diagnosed disease high profile variability * no strict limits for clear positive/negative disease detection C7250 J. LaBaer et al., J. Proteome Res., 4 (4) 1053-1059 (2005). M. Ehmann et al., Pancreas, 34 (2) 205-214 (2007). C7250 MS-based approaches for biomarker searching Pattern profiling direct MS analysis (MALDI MS, SELDI MS) comparison of peptide (protein) profile of sample „healthy“ vs. patient (bez identifikace, statistická analýza) early disease detection biomarker identification Separation GE. LC comparison of protein (peptide) content of sample „healthy“ vs patient MS MS/MS identification of differentialy regulated proteins specific antibody Immunodetection SRM, SWATH/Protein arrays biomarker specifity!!! C7250 Glycan profiling and structural analysis of glycans NSCLC - Bronchoalveolar Carcinoma Bronchoalveolar Adenocarcinoma Large Cell Carcinoma 0 1 2 3 4 4 x10 2000 2500 3000 3500 4000 m/z 0 2 4 6 4 x10 0 0.5 1.0 1.5 4 x10 3x 3x 4x 4x 6x 5x 5x 3x 6x 4x 5x 3x 5x 3x 4x 3x 4x 5x 3x GlcNAc; Fuc Gal; Man; MALDI-TOF-MS spectra of N-glycans after desialylation 18 Lattová E., J. Proteome Res., 15 (8), 2777-2786 (2016) C7250 https://s-media-cache-ak0.pinimg.com/736x/79/f8/39/79f8397406fc725e32e659addc624213.jpg Comparison of human cancer cell line proteome and transcriptome C7250 126 frakcí 72 frakcí N. Nagaraj et al., Mol. Syst. Biol., 7, 548 (2011). 10 mg proteins Protein fractionation Peptide fractionation pic11840 Relative quantification by MS 100% * isotopically labeled tag approaches (comparison of limited number of samples, up to 10) protein quantification Targeted quantification of selected proteins by MS C7250 * label-free approaches (comparison of unlimited number of samples, lower accuracy) replica 1 replica 2 replica 3 * MRM, SWATH C7250 B Characterization of proteome changes differential (expression) proteomics cooperation with Department of Biochemistry, FS MU P. Bouchal et al., Proteomics 2006, 6, 4278–4285. Acidithiobacilus ferrooxidans grown on ferrous iron (A) and elemental sulfur (B) image analysis of 2-D gels LC-MS/MS of selected spots with different intensity identification (MS) separated from quantification (spot intensity on gel) mixed spots control infected gut wall sample protein isolates tryptic digestion reductive alkylation LC-MS/MS data processing C7250 Search for marker proteins for chicken salmonella infection relative quantification * identification more than 2300 protein * quantification for more than 1900 cooperation with VRI Brno Matulova M. et al., Vet. Res., 44:37 (2013) Accession Description 363741657 PREDICTED: syntenin-2-like [Gallus gallus] 41.032 118095649 PREDICTED: beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase 3 [Gallus gallus] 34.036 4927286 alpha enolase [Bos taurus] 33.575 112491068 Chain A, Crystal Structure Of A M-Loop Deletion Variant Of Ment In The Cleaved Conformation 30.221 56118294 ribonuclease homolog precursor [Gallus gallus] 25.497 363741459 PREDICTED: protein-glutamine gamma-glutamyltransferase E [Gallus gallus] 24.786 infected/ control confirmation by real-time PCR clarifying of mechanisms of molecular processes search for marker proteins for early detection C7250 C7250 Quantification of enterotoxins targeted analysis of selected protein MRM * selection of peptides suitable for MRM * absolute quantification by AQUA peptides result- amount/concentration of given protein in sample SWATH MS Q-TOF, MS/MS < 10 ppm Retention time m/z * classic DB searching not possible * comparison with libraries of ref. MS/MS spectra * Relative and absolute quantification all MS/MS spectrum Gillet et al, MCP, 11, 1-17 (2012) C7250 Liu et al, Expert Rev. Mol. Diagn., 13(8), 811 (2013) Shot gun vs SRM vs SWATH C7250 Liu et al, Expert Rev. Mol. Diagn., 13(8), 811 (2013) C7250 Liu et al, Expert Rev. Mol. Diagn., 13(8), 811 (2013) C7250 a62 Chen et al., J. Chromatogr. B, 879, 25 (2011) Phosphoproteome analysis – four fractionation approaches C7250 ERLIC - Electrostatic Repulsion-Hydrophilic Interaction Chromatography Each method – over 4000 phoshopeptides In total – 9069 phosphopeptides – 9463 sites / 3260 proteins Chen et al., J. Chromatogr. B, 879, 25 (2011) C7250 Phosphoproteome analysis – four fractionation approaches C7250 Phosphoproteome analysis– quality and quantity Huang et al., J. Proteomics, 106, 125 (2014) Porcine muscle Postmortem changes 160 phosphoproteins with 784 sites identified 184 phosphorylation sites on 93 proteins had their phosphorylation levels significantly changed. Huang et al., J. Proteomics, 106, 125 (2014) C7250 Phosphoproteome analysis– quality Comparison of identified peptides in replicas The panels show the phenotypic phosphoproteome comparison organized by GO biological process for mitotic (left) and S phase (right) cells. Proteins involved in metabolic processes have high-occupancy phosphorylation sites during mitosis, but low-occupancy sites during S phase (color scale: yellow, high overrepresentation; dark blue, high underrepresentation). Olsen J.V. et al., Sci. Signal., 3 (104) ra3 (2010) * * quantified 6027 proteins * quantified 20,443 unique phosphorylation sites Phosphoproteome analysis CG010 - HELA cells - SILAC labeling - TiO2 enrichment - LC-MS/MS (Orbitrap) C7250 * to establish a set of methods - HDAC Fluorimetric Cellular Activity Assay Kit - MALDI-MS of N-terminal part of histones (after Glu-C digestion) - AUT-AU 2-D GE combined with LC-MS/MS analysis Characterization of effect of histone deacetylase inhibitors Valproic acid Entinostat Total HDAC inhibiton effect Činčárová et al, Mol. Biosyst. (2012) B-CLL MEC-1 cells C7250 Valproic acid Entinostat MALDI-MS of Histone H4 acetylated forms N-terminal fragment (1-53 AA) control A E 0 4 acetylations Characterization of effect of histone deacetylase inhibitors C7250 Valproic acid Entinostat Changes of particular H4 acetylated forms vs inhibitor concentration (24h treatment) A E 0 4 acetylations VPA [mM] Enti [mM] Characterization of effect of histone deacetylase inhibitors C7250 AUT-AU 2-D GE of histone extract Characterization of effect of histone deacetylase inhibitors C7250 AUT-AU 2-D GE of histone extract ctrl ctrl VPA ENTI A E 0 4 acetylations Characterization of effect of histone deacetylase inhibitors C7250 MALDI-MS 2-D AUT-AU GE peak intensity spot intensity Incubation time (hr) Changes of histone H4 acetylated forms in dependence on incubation time (2 mM VPA) Characterization of effect of histone deacetylase inhibitors 43 histone extraction TCA precipitation derivatization at protein level tryptic digestion derivatization at peptide level LC-MS/MS Characterization of Post-Translational Modifications of Histones e.g. in Sidoli S. et al., J. Vis. Exp. 111:e54112 (2016). 44 Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones human recombinant histone standards human histone extract after TCA precipitation re-dissolved in MilliQ water A. thaliana histone extract after TCA precipitation re-dissolved in MilliQ water A. thaliana histone extract after TCA precipitation re-dissolved in SDS A. thaliana histone extract in sulfuric acid diluted in MilliQ water Ledvinova D.. et al, Front. Plant Sci., 9, article 1373 (2018) C7250 45 Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones Ledvinova D.. et al, Front. Plant Sci., 9, article 1373 (2018) C7250 single mixed 46 Inter-individual variations of Histone Modification Patterns cooperation with Prof. Fajkus group, CEITEC-MU two Arabidopsis thaliana ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) grown from seeds collected from a single parent plant (Single) and a set of parent plants (Mixed). Brabencova S. et al, Front. Plant Sci., 8, article 2084 (2017) C7250 a61 * > 80% proteins is functional only as a part of complex * ~ 10000 types of interactions Aloy P., Russell R. B.: Nat. Biotechnol. 22 (10), 1317-1321 (2004) Charakterization of protein complexes functional proteomics complex purification sample (cells) MS/MS analysis confirmation of true interaction partners * search for new intraction partners * study of „whole“ complex C7250 T. Köcher, G. Superti-Furga: Nat. Methods, 4(10), 807-815 (2007). Purification of protein complexes in vivo expression of bait protein with a tag high degree of complex purity loss of weakly binded interaction partners C7250 Gavin A.-C. et al.: Nature, 440 (30), 631-636. • identification of individual complex members including their PTMs • confirmation of interaction partners (exclusion of nonspecific interactors) • determination of complex stoichiometry • determination of 3D structure of complex (cross-linking) MS capabilities in protein complex analysis C7250 ctrl complex Identification of individual interaction partners C7250 digestion in gel LC-MSMS ctrl complex digestion in solution LC-MSMS sample prep possibility to label samples by tags for relative quantification and to pool the samples for further processing Confirmation of true interaction partners W. Yang et al., Proteomics 2008, 8, 832–851 non-specific interaction 1:1 C7250 a56 8M slina pH3,9-5,1 13-2-09 Sequence confirmation and determination of OBP protein isoforms de novo sequencing Obp3A Obp3B Obp2 Myodes glareolus * saliva * 2D gel electrophoresis * MS/MS of selected spots Stopková R., Zdráhal Z., Ryba Š. et al, BMC Genomics 2010, 11:45 cooperation with prof. Stopka FS CU, Prague * unknown genome * no antibodies C7250 C7250 RNA analysis (FS CU Prague) proteomic analysis (FS CU/Proteomics CF) RNA isolation cDNA synthesis PCR with Aphrodisin primers (hamster) purification cloning sequencing initial aminoacid sequence protein isolation 2D GE in-gel digestion MALDI-MS/MS de novo corrected AA sequence (Blast, new proteins) database of OBP protein sequences identification of protein isoforms Sequence confirmation and determination of OBP protein isoforms m/z 3079.4 DAELEGTWYTTAIAADNVDTIEEEGPLR MALDI-MS/MS original sequence - QAELEGKWVTTAIAADNIDTIEEEGPMR (OBP3) DAELEGTWYTTAIAADNVDTIEEEGPLR HAELEGTWYTTAIAADNVDTIEEEGPLR MALDI-MS/MS a LC-MS/MS manual spectra interpretation C7250 Sequence confirmation and determination of OBP protein isoforms a75 MS Imaging C7250 samples: * fresh frozen tissue sections * individual cells or clusters of cells isolated by laser-capture microdissection or contact blotting of a tissue on a membrane target. MALDI-MS imaging MS analysis * scanning of sample area point by point * image corresponds to planar distribution of individual m/z distribution of peptides (proteins, lipids,...) * C7250 MALDI-MS imaging M. Stoeckli, T.B. Farmer, R.M. Caprioli: J Am Soc Mass Spectrom, 10, 67–71 (1999) C7250 MALDI-MS imaging Chaurand et al:Anal. Chem.2004, 76,1145-1155 C7250 http://dghxm7w6km1w9.cloudfront.net/content/ajpendo/302/8/E1016/F5.large.jpg?width=800&height=600&c arousel=1 Degradation of Ang III and Ang-(1–7) in mouse kidney sections. C7250 Grobe N. et al, Am. J. Physiol. Endocrinol. Metab., 302 (8), E1016 (2012) The end