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 Assessment of products of synthesis MALDI-MS C7250 MW(adukt) = 250 Počet navázaných aduktů N = 12 20000 40000 60000 80000 100000 120000 140000 m/z 0 200 400 600 800 1000 1200 1400 a.i. /D=/Data/Zbynek/020205/BSA/2Lin/pdata/1 Administrator Fri Feb 22 13:59:04 2002 Modified BSA vs. std. BSA (» 5 pmol) mass diff. cca 3 kDa MW(adukt) = 250 Number of binded molecules N = 12 C7250 MALDI - MS Assessment of reaction course 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 10 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 N-Glycan profiling of lung adenocarcinoma in patients at different stages of disease GlcNAc; Fuc Gal; Man; MALDI-TOF-MS spectra of N-glycans after desialylation Lattová E. et al., Modern Pathology (2020) 33:1146–1156 C7250 Detail of MALDI-MS spectra (m/z range of 2650-4150): a) P1 (stage IB/grade1) b) P7 (stage IIA/grade4) c) P13 (stage IIIA/grade2) https://s-media-cache-ak0.pinimg.com/736x/79/f8/39/79f8397406fc725e32e659addc624213.jpg 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 (SRM), PRM 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 20 Hafidh S. et al., Plant Physiol., 178 (1), 258-282 (2018) the three types of mRNA-containing ribonucleoprotein particles: •POL - translating polysomes •RNP - free ribonuclear particles •EPP - long-term storage EDTA/puromycin-resistant particles six developmental stages •9317 protein groups identified across all samples and replicates •2,089 for quantitative analyses (only proteins identified by five or more peptides in all biological and technical replicates of the particular sample were considered as reliably present). Characterization of the extent and dynamics of translational regulation during tobacco male gametophyte development and the subsequent functional progamic phase 37 21 Characterization of the extent and dynamics of translational regulation during tobacco male gametophyte development and the subsequent functional progamic phase Hafidh S. et al., Plant Physiol., 178 (1), 258-282 (2018) Overall abundance of PABP transcripts Dynamics of seven protein groups associated with PABPs affect mRNA cellular fate, metabolism, translation, and storage The quantitative and condition-dependent Escherichia coli proteome Schmidt A., Nature Biotechnol., 34 (2016), 104 – 110 22 different growth conditions in biological triplicates. (i)growth on minimal media with an excess of different carbon and energy sources (ii)growth in glucose-limited chemostat cultures with varying growth rates, (iii)growth on glucose excess with different stress conditions, (iv)growth on complex medium, and (v)1 and 3 d into stationary phase. cellular protein concentrations for 55% of predicted E. coli genes (>2,300 proteins) 41 proteins related to glycolytic pathway, tricarboxylic acid cycle enzymes and others was selected to absolute quantification The concentration range of the 41 proteins covered more than four orders of magnitudes ranging from around 92,000 to only 2 copies per cell. 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 C7250 Targeted analysis of selected protein/PTM Step1: 1. Biological samples include body fluids (such as blood or saliva), cell lysate, and tissue. 2. PTM proteins and protein isoforms are extracted from cell lysate and tissue. 3. Protein enrichment methods are applied for less abundant proteins. 4. PTMs and protein isoforms are digested by enzymes, including trypsin, Lys-C, etc. 5. After digestion, modified, unmodified peptides, and peptides representing the whole protein are selected. Step2: 1. Isotope-labeled peptides are synthesized to serve as internal standards for the post-translationally modified peptides, unmodified peptides, and peptides representing the whole protein. 2. Mass spectrometer parameters are optimized by using the synthetic peptides. 3. Isotopic labeled peptides are internal standards and spiked in the samples. PTMs and isoforms are quantified by SRM or MS3 assay. Schematic workflow of a constrained SRM assay Liu X., et al., Methods 61 (2013) 304–312 Quantitative assays for ppErk1. (A) Erk1 phosphorylation was measured by a constrained SRM assay in trypsin-digested extracts from EGF-stimulated vascular smooth muscle cells. Extracted ion chromatograms are presented with the area under the curve (1000) displayed in the top right corner. Heavy isotope-labeled internal standards were utilized for peak identification and normalization. SRM C7250 SRM and western blot assays show a similar time-course of Erk1 phosphorylation in response to EGF. IADPEHDHTGFLTEYVATR IADPEHDHTGFLTEYVATR – 2x Phospho Liu X., et al., Methods 61 (2013) 304–312 Targeted analysis of selected protein/PTM 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 37 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). 38 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 39 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 40 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 Kucharikova et al, Mol. Cell. Proteomics, 20, 100114 (2021) Improvement of separation of histone peptide modified forms novel derivatization agent Histone H4 4-17 GKGGKGLGKGGAK Histone extracts from MEC1 cell line. ~ 2x more forms C7250 Assessment of effect of histone deacetylase inhibitors HDAC inhibitors – potential for cancer treatment Kucharikova et al, Mol. Cell. Proteomics, 20, 100114 (2021) C7250 Stukalov A. et al., bioRxiv: https://doi.org/10.1101/2020.06.17.156455 Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV C7250 (a)Time-resolved profiling of SARSCoV-2 infection by multiple -omics methods. The plot shows normalized MS intensities of three SARS-CoV-2 viral proteins over time and overview of identified and regulated distinct transcripts, proteins, ubiquitination and phosphorylation sites, using data independent (DIA) or dependent (DDA) acquisition methods GG enrichment TiO2 enrichment 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 BioID unique method to screen for physiologically relevant protein interactions that occur in living cells. Choi-Rhee E, Schulman H, Cronan JE. Protein Sci. 2004;13(11):3043–50 Enzyme - BirA - the biotin protein ligase - of Escherichia coli biotinylates only a single cellular protein. a mutant BirA attaches biotin to a large number of cellular proteins in vivo Enzyme-catalyzed proximity labeling The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Biotinylation is a rare protein modification in nature, it enables selective isolation and identification with standard biotin‐affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. BioID Roux KJ, Kim DI, Burke B. Curr Protoc Protein Sci. 2013;74:Unit 19 23. Enzyme-catalyzed proximity labeling Roux KJ, Kim DI, Raida M, Burke B. J Cell Biol. 2012;196(6):801–10 BioID Enzyme-catalyzed proximity labeling Enzyme-catalyzed proximity labeling TurboID/miniTurbo •multiply mutated BirA •higher efficiency of labeling than BioID or BioID2 •faster kinetics Branon T., C. et al., Nat Biotechnol. 2018 October ; 36(9): 880–887 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) bruker.com Liu et al, Expert Rev. Mol. Diagn., 13(8), 811 (2013) C7250 H. Brinkerhoff et al., Science 10.1126/science.abl4381 (2021) Fig. 1. Reading peptides with a nanopore. A) The DNA-peptide conjugate consists of a peptide (pink) attached via a click linker (green) to an ssDNA strand (black). This DNA-peptide conjugate is extended with a typical nanopore adaptor comprised of an extender that acts as a site for helicase loading (blue) and a complementary oligo with a 3′ cholesterol modification (gold). B) The cholesterol associates with the bilayer as shown in (a), increasing the concentration of analyte near the pore. The complementary oligo blocks the helicase, until it is pulled into the pore (b), causing the complementary strand to be sheared off (c), whereupon the helicase starts to step along DNA. A DNA-peptide conjugate was pulled through the biological nanopore MspA by the DNA helicase Hel308. Reading the ion current signal through the nanopore enabled discrimination of single–amino acid substitutions in single reads. Nanopore peptide sequencing C7250 H. Brinkerhoff et al., Science 10.1126/science.abl4381 (2021) Nanopore peptide sequencing C7250 (C) As the helicase walks along the DNA, it pulls it up through the pore, resulting in (a) a read of the DNA portion followed by (b) a read of the attached peptide. (D) Typical nanopore read of a DNA-peptide conjugate (black), displaying step-like ion currents (identified in red). The asterisks * indicate a spurious level not observed in most reads and therefore omitted from further analysis. The dagger † indicates a helicase backstep. (E) Consensus sequence of ion current steps (red), which for the DNA section is closely matched by the predicted DNA sequence (blue). The linker and peptide sections are identified by counting half-nucleotide steps over the known structural length of the linker. Error bars in the measured ion current levels are errors in the mean value, often too small to see. The end