ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT. financed by the program

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1 TÁMOP C-13/1/KONV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: OKTÓBER 04. / OCTOBER 04, 2017 Helyszín / Place: MTA SZBK BIOFIZIKAI INTÉZET, TANÁCSTEREM / LECTURE ROOM, INST. OF BIOPHYSICS, BIOLOGICAL RESEARCH CENTRE SZEGED, TEMESVÁRI KRT. 62. Az előadás címe / Title of the presentation: MASS SPECTROMETRY BASED PROTEOMICS Előadó / Speaker: ZSUZSANNA DARULA Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB

2 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP C-13/1/KONV Mass spectrometry based proteomics Zsuzsanna Darula Institute of Biochemistry October 04, 2017 Proteomics: large scale study of proteins Proteome: the complete set of proteins expressed by a cell / tissue / organism at a given time under defined conditions Separation / fractionation of protein samples 1.: gel electrophoresis 1D: SDS PAGE (size separation, limited resolution, low complexity samples) 2D: IEF SDS PAGE isoelectric point size resolving power: a few thousand proteins/gel limitations: membrane proteins highly acidic / basic proteins very small/ large proteins 16 BAC/SDS PAGE size separation in both dimensions, limited resolution Native gel SDS PAGE analysis of protein complexes: isolation of complex in 1 st dimension, separation of components in the second 1

3 Separation / fractionation of protein samples 2.: Chromatography Type Abbr. Principle of separation Size exclusion chromatography SEC Differences in size and shape Ion exchange chromatography IEC Electrostatic interactions (pk a, pk b ) Normal phase chromatography Hydrophilic interaction chromatography Reversed phase chromatography Hydrophobic interaction chromatography NPC HILIC RPC HIC Polar interactions Dispersive interactions Affinity chromatography AC Specific interactions Identification of separated proteins 1. Edman sequencing REAGENT H 2N Amino Acid 1 AA 2 AA 3 AA 4 AA 5 REAGENT Amino Acid 1 AA 2 AA 3 AA 4 AA 5 N reagent Phenylisothiocyanate (PITC) C S ph:8 H 2N HN R O Peptide first amino acid unit H, heat Amino Acid 1 REAGENT H 2N AA 2 AA 3 AA 4 AA 5 S N NH R H 2N Peptide HPLC O Phenylthiohydantoin amino acid reagent product requires pure protein requires free protein N terminus (acetylation?) ~30 (max 50 60) AA can be determined sample requirements: 10 pmol slow (~ 1h / AA) ~ isobaricaa s can be distinguished (I/L; Q/K) Identification of separated proteins 2. Western blot sensitive specific (semi)quantitative requires antibody foreknowledge of protein of interest is required expensive 2

4 Identification of separated proteins 3. Mass Spectrometry (MS) Sensitive (fmol/amol range) Quick No antibody or external standards required Amenable with mixtures blocked peptides modified peptides (PTM analysis) Problems with distinguishing isobaric AA s (Leu/Ile (and Gln/Lys)) Mass Spectrometry (MS) Determination of m/z value of gas phase ions (mass to charge ration, m: mass, z: charge) VACUUM SYSTEM spectrum sample ION SOURCE MASS ANALYZER DETECTOR Int. signal Formation of ions Separation of ions Detection of ions m/z Ion source generation of gas phase ions soft ionization techniques MALDI: matrix assisted laser desorption ionization, singly charged ions ESI: electrospray ionization, multiply charged ions Mass analyzer separation of ions according to m/z defines performance of the mass spectrometer Sensitivity Resolution Mass accuracy (absolute / relative (ppm!!!)) Linear dynamic range Speed Mass range analyzers used in proteomics: Quadrupole, Ion trap, flight tube (TOF), FT ICR, Orbitrap (alone or in combo) 3

5 Theorethical peak shape of peptide m/z: 1000 (z=1) at different resolution R=500 R=1000 R=2000 R=10000 Monoisotopic mass only 12 C, 1 H, 14 N, 16 O (and 32 S) x 13 C(and ) 2x 13 C(and ) Element C H O N S Mass number Natural occurrence % Monoisotopic peak is ALWAYS the first in the isotope cluster but not necessarily the most abundant! m/z: 300 m/z: 1800 m/z: 3000 m/z:

6 Isotope spacing enables charge determination _48_6PM # 1907 RT: AV: 1 NL: 1.67E5 T: FTMS p NSI Full ms [ ] Relative Abundance Δ m/z: 1 z= Δ m/z: 1/ Δ m/z: 1/3 z= m/z MS analysis of proteins Top down Intact proteins are investigated Bottom up Peptides generated from proteins are investigated Peptide mass fingerprinting (For low complexity samples) Tandem mass spectrometry (For high(er) complexity samples) TOP DOWN APPROACH molecular weight determination of intact proteins fragmentation of intact isolated proteins (ETD, ECD, CID, HCD) degradation products sequence variants combinations of post translational modifications positions of disulfide bridges techniqually challenging lower MW proteins limited sample complexity high resolution (R> !), special instrument required 5

7 Bottom up approach Generation of peptides from proteins Enzymatically Endopeptidase Specificity ph range Trypsin R, K Chymotrypsin Y, F, W, L Glu C E, D Asp N D Arg C R Lys C K Lys N N Chemically Cyanogen bromide (Met (Trp )) Acidic hydrolysis (Asp, (Glu )) Enzymatic protocols most often use trypsin Cleaves after Lys and Arg (except the next AA is a Pro) Provides at least one basic amino acid per peptide (facilitates ion generation for MS) Statistically the size of the tryptic peptides are perfect for m/z range of analyzers (~10% of the AA content is R or K) Cheap, reliable, known sequence Modified trypsin is available commercially ( autolysis) Peptide mass fingerprinting (PMF) 2D gel electrophoresis reduction alkylation trypsin peptide extraction desalting MALDI TOF Database Search Int. Protein list m/z MS compatible staining Reduction: DTT (dithiothreitol), mercaptoethanol, TCEP (tris(2 carboxyethyl)phosphine) Alkylation: IAM (iodoacetamide), IAA (iodoacetic acid), NEM (N Ethylmaleimide), 4 vinylpyridine 6

8 MALDI Ion Source (Matrix Assisted Laser Desorption Ionization) Laser beam hν Target Plate Desorption Desolvation Ionization to Analyzer Co crystallized matrix and analyte molecules Protein or peptide analyte Acidic matrix molecule O Commonly used matrices O O HO OH OH H 3CO OH OH HO CN HO OCH 3 DHB CHCA SA 2,5 DihydroxyBenzoic Acid α Cyano 4 HydroxyCinnamic Acid Sinapinic Acid TOF Analyzer (time of flight) Repeller Laser beam Detector Grid ( pole) Signal Int. Spectrum Plate Analytes with different m/z values (different velocity different amount of time to reach the detector) m/z generated ions are accelerated by applied electric field ions of same charge have the same kinetic energy velocity of the ions depends on their mass to charge ratio time to reach the detector is measured, m/z calculated m z 2 2 t V 2 L or t m z MALDI TOF spectrum of tryptic digest of a ~70 kda protein a.i m /z 7

9 Search engines: Mascot, Protein Prospector 8

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11 100 % sequence coverage? peptide m/z out of detection range (short and long peptides) poor recovery of hydrophobic peptides (extraction from gel) loss of hydrophilic peptides (desalting) ion suppression (MS analysis) incorrect sequence in database protein processing (signal peptide, propeptide) MALDI-TOF MS, without fractionation 3 different components T T PMF SUMMARY Protein is cleaved into peptides in a specific manner (usually using trypsin) The m/z value of the resulting peptides is determined using mass spectrometry (typically MALDI TOF) Protein is identified by database search by comparing experimentally determined peptide m/z s to theoretical ones generated in silico from proteins present in database Applicable to low complexity samples (a few protein/sample) 10

12 For more complex peptide mixtures: MS/MS based protein ID Ion source - ionization Analyzer 1 ionization selection fragmentation ionization Analyzer 2 m/z separation Detector detection sample Int. Tandem in space (two separate analyzers, Q TOF, Q TRAP, IT Orbitrap ) Tandem in time (single analyzer, ion traps) m/z MS/MS (fragmentation) spectrum Electrospray ionization (ESI) (heated) capillary e - to the analyzer - High voltage Sample nebulized to fine aerosol Size of sample droplets is reduced by applied electric field and heat (desolvation) Droplets explode when electrostatic repulsion overcomes surface tension Finally completely desolved ions enter the mass analyzer Multiply charged ions are formed On line coupling to HPLC: LC MS/MS FRAGMENTATION Energy based Energy is put into the peptide (weakest bonds break) Collision Induced Dissociation (CID/CAD, HCD) Infra Red MultiPhoton Dissociation (IRMPD) Radical based Electrons create unstable radical ions that spontaneously fragment at sites of electron capture Electron Capture Dissociation (ECD) Electron Transfer Dissociation (ETD) 11

13 Sequence ions are formed by fragmentation of the peptide backbone: SPECIFIC TO PEPTIDE SEQUENCE! x 3 y 3 z 3 x 2 y 2 z 2 x 1 y 1 z 1 H 2 N R 1 O R 2 N H H N O R 3 O R 4 N H O OH a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3 Collision induced dissociation (CID) 1. Sequence ions H 2 S A M P L E R OH y 6 y 5 y 4 y 3 y 2 y 1 b 1 b 2 b 3 b 4 b 5 b 6 b fragment ions y fragment ions 6 H S A M P L E H 2 R OH 1 5 H S A M P L H 2 E R OH 2 4 H S A M P H 2 L E R OH H H H S A M S A S H 2 P L E R OH H 2 M P L E R OH H A M P L E R OH Instrument dependent fragmentation Fragment ions generated by CID: 1. Sequence ions (a, b, y) 2. Internal fragments (if multiple fragmentation) 3. Satellite ions (water and NH 3 loss of fragment ions) 4. Immonium ions (info on amino acid content) 12

14 LC MS/MS: data dependent analysis RT : Relative Abundance BPI Time (min) NL: 2.72E7 Base Peak F: ms MS _27 Ions eluting from HPLC _27 #8826 RT: AV: 1 NL: 4.20E6 T: FTMS p NSI Full ms [ ] Relative Abundance MS z= z= z= z= m/z Ions detected in Orbitrap analyzer at 41.43min _27 #8827 RT: AV: 1 NL: 2.90E5 T: ITMS c NSI d w Full ms @cid35.00 [ ] Relative Abundance MS / MS m/z MS/MS spectrum of precursor ion m/z: (2), fragmented and measured in ion trap 1. Generation of peak list (txt, mgf, dta ): List of all MS/MS data acquired during an LC MS/MS experiment: For each MS/MS spectra: precursor m/z, precursor charge state list of m/z and intensity values for observed fragment ions 2. Database search All MS/MS spectra is searched individually, results are given as proteins listing peptides assigned to them Search engine (Mascot, Protein Prospector, OMSSA, Sequest, Proteome Discoverer, Byonic ) 1. defines peptide candidates with the same (theoretical) m/z that is observed for the precursor (in silico digestion of proteins in the database) 2. compares observed fragment ion list to theoretical fragment ion list of peptide candidates (instrument dependent fragmentation!!!) 3. assigns a score and / or a probability value (expectation value, E value) to peptide matches for deciding the goodness of identifications 4. lists the highest scored match(es) for each peptide ID meeting acceptance criteria 13

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16 One peptide to multiple proteins? Homology Between species Protein families within the same species Different isoforms of the same protein Coincidence Sample Gene Acc.No. Protein Protein MW Unique Peptides Coverage 1 At3g02090 Q42290 mitochondrial-processing peptidase % beta subunit, (MPP beta) 1 At3g16480 O04308 mitochondrial-processing peptidase 54 1 a 3.00% alpha subunit 2, (MPP alpha-2) 1 At2g07727 P42792 Cytochrome b (MTCYB) (COB) b 3.30% (CYTB) 2 At1g51980 Q9ZU25 mitochondrial-processing peptidase 54 c % alpha subunit 1 (MPP Alpha-1) 2 At3g16480 O04308 mitochondrial-processing peptidase 54 c 1 c (9) 16.40% alpha subunit 2, (MPP alpha-2) 3 At5g40810 Q0WNJ4 Cytochrome c1 (CYC1-2) % 4 At5g13430 Q9LYR3 Ubiquinol--cytochrome-c reductase % (REISKE subunit) 5 At4g32470 Q9SUU5 Ubiquinol-cytochrome C reductase 14.5 d % complex 14 kda protein 5 At5g25450 Q3E953 Ubiquinol-cytochrome C reductase complex 14 kda protein 14.6 d 1 d (2) 18.00% 15

17 All MS/MS data assigned to peptides? NO non peptide components (salts, detergents, derivatizing agents ) incomplete reduction / alkylation upon sample preparation post translational modifications side reactions during sample preparation cyclization (N terminal Gln, Cys(CAM)) methylation (Glu, Asp) oxidation (Met, Trp, Cys, Tyr) carbamylation (N term, Lys) carbamidomethylation (N term, Lys, Met, His, Asp) deamidation (Asn, Gln) S acrylamide formation (Cys) formation of alkali metal adducts of peptides nonspecific cleavages incomplete digestion (number of missed cleavages?) multiple peptides selected and fragmented in source fragmentation resulting in nonspecific peptides in source water loss (Ser, Thr, Asp, Glu) short peptides may not yield enough fragments for confident ID low quality spectra incorrect monoisotopic m/z incorrect charge state peptides/proteins not present in database 16

18 % % Zs. Darula October 4, 2017 Post translational modifications 1. proteolytic cleavages N terminal Met cleavage signal peptide propeptide chemical group acetylation, phosphorylation, glycosylation, ubiquitination, sumoylation, lipidation... intra or inter peptidic linkages disulfide bonds... Post translational modifications 2. (if) reflected in the molecular weight of the protein and corresponding peptide: amenable to MS usually substoichiometric: requires enrichment of modified peptides prior to MS modified peptides may feature similar or different fragmentation pattern: alternative fragmentation techniques (e.g. ETD for glycopeptides) characteristic fragmentation: pinpointing modified peptides (carbohydrate oxonium ions for glycopeptides, neutral loss ions for phosphopeptides / Met oxidized peptides) Phosphopeptides fragment similarly to unmodified peptides (CID) Characteristic 98 Da neutral loss (phosphate) HVG _szbk_01 57 (21.531) Cn (Top,4, Ar); Sm (Mn, 2x1.00); Sb (1,40.00 ); Cm (57:58) : TOF MSMS ES 7.71e phosphopeptide peptide _szbk_02 5 (21.980) Cn (Top,4, Ar); Sm (Mn, 2x1.00); Sb (1,40.00 ); Cm (5:6) Da Da 98 Da Da Da Da Da : TOF MSMS ES m/z 17

19 Glycopeptides show different fragmentation than unmodified peptides (CID) 100 y Relative Abundance TPIVGQPSIPGGPVR y y b 6 b b y y 8 * b (2) y 5 y8 y 10 y m/z _02 #2601 RT: AV: 1 NL: 1.53E4 T: ITMS p NSI t d Full ms @cid35.00 [ ] Relative Abundance Sialic acid related oxonium ions Carbohydrate loss related fragment ions Same peptide glycosylated (HexNAcHexSA) m/z Quantitative proteomics 1. Gel based 2D gel electrophoresis time consuming, labor intensive limited dynamic range not suitable for LMW ( 15kDa) and HMW (>150 kda) protein hydrophobic (e.g. membrane) proteins insoluble proteins needs min. 100 g total protein/gel 3 replicates / sample Improvements more sensitive staining large format higher resolving gel sample prefractionation DIGE (Difference Gel Electrophoresis, fluorescent labeling) Quantitative proteomics 2. Gel free MS based Stable isotopic labeling ( 2 H, 13 C, 15 N, 18 O) Chemical labeling ICAT (isotope coded affinity tag, Cys) itraq, TMT (Multiplexed isobaric tagging technology, Lys/peptide N term) ICPL (isotope coded protein label) Formaldehyde NaBH 3 CN (peptide N term, Lys) Enzymatic labeling 16 O/ 18 O exchange catalyzed by trypsin (peptide C term) Metabolic labeling SILAC (Stable isotope labeling with amino acids in cell culture) 15 N or 13 C labeling (complete or partial) Label free LC MS quantification comparison of ion signal intensities (spectral counting), or chromatographic peak areas (MS E ) 18

20 Cell/ tissue SILAC ICAT N/C term. itraq, TMT Internal Standards Protein Peptide MS Thank you for your attention! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practiceoriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMOP C-13/1/KONV project. 19