PROTEOMICS (Mass spectrometry in Biochemistry)
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1 PROTEOMICS (Mass spectrometry in Biochemistry) A lecture combined with laboratory experiments Friedrich Altmann / Department für Chemie, Universität für Bodenkultur Wien
2 Genomics - Why? Why in English? Why should you attend? Why almost no advertising?
3 Genomics - What is the molecular difference of cells / tissues in different situations? Genomics: quantitative analysis of transcribed genes = mrna Splicing, mrna stability, translation efficiency, folding efficiency, protein stability and turnover, protein modifications : quantitative analysis of proteins present
4 A popular approach: Proteins are separated by 2D electrophoresis several hundred spots, even more with prefractionation IEF (ph) SDS (Size)
5 Quantitative 2D electrophoresis healthy control tumour tissue?
6 Chapter 1: Mass spectrometry of small molecules Why mass spectrometry Electron impact ionization GC-MS
7 Identification of small molecules Specific reactions Spectroscopy (UV/VIS, IR, NMR) Chromatography (TLC, RP-HPLC) Mass spectrometry -> Hyphenated techniques
8 Identification of small molecules In very complex mixtures and for minor compounds, a clear peak cannot always be obtained. Abs Dexamethazone in urine UV-detection time Dexamethasone, a corticosteroid, is used to replace this chemical when the body does not make enough of it. It relieves inflammation (swelling, heat, redness, and pain) and is used to treat certain forms of arthritis; severe allergies; asthma etc.
9 Identification of small molecules Non-selective detection: UV or MS total ion current Abs Dexamethazone in urine Selective detection: MS or even better MS/MS here: MS/MS transitions from 393 to 121,147 and 237 Similar: Pesticides, mycotoxins, illegal drugs etc etc.
10 Mass spectrometry is the determination of the mass per charge (m/z) of an ion in vacuum
11 Mass spectrometry 1. Ionization of analyte 2. Removal of neutral molecules 3. Determination of masses of ions
12 Mass spectrometry of volatile molecules Analyte in the gas phase is transferred to vacuum Bombarded with accelerated electrons Electron impact ionization (EI)
13 Mass spectrometry of volatile molecules The usual ionization is EI at 70 ev. Thereby more energy is transferred to the molecule than necessary for ionization. Excess energy leads to fragmentation of analyte Fragment spectrum is fingerprint of molecule.
14 Mass spectrometry of volatile molecules EI ionization: Fast e - removes a normal e - from analyte. As M * is unstable, fragments F * 1, F * 2, F * 3,... and N 0 1, N 0 2 are formed M = 334 Intensity (%) m / z
15 Mass spectrometry of volatile molecules Analyte in the gas phase is transferred to vacuum Collided with ionized reactant gas Chemical ionization (CI) Gentle ionization, gives strong molecular ion ( M * )
16 Mass spectrometry of volatile molecules EI ionization (and also CI) can be coupled with gas chromatography. GC / MS ( GLC / MS ) For volatile molecules or molecules made volatile by derivatisation (acetylation, trimethylsilylation, methylation...) FAMEs (fatty acid methyl esters), TMS-everything
17 GC-MS Total ion current (TIC) chromatogram 5-Araf 983 t-araf 698 3,5-Araf 2,5-Araf ,6-Galp scan number (1 scan = 2 sec)
18 ,5-Ara Spectra 3,5-Ara m / z m / z 5-Araf Gal TIC Chromatogram t-araf ,5-Araf ,5-Araf ,6-Galp m / z scan number (1 scan = 2 sec)
19 For volatile molecules: 1. Electron Impact Ionisation 2. Chemical Ionisation For non-volatile molecules: 3. historic attempts Ionisation methods 4. Atmospheric Pressure Ionisation (API) 4a. Electrospray Ionisation (ESI) 4b. Atmospheric Pressure Chemical Ionisation (APCI) 5. Matrix Assisted Laser Desorption Ionisation (MALDI)
20 Chapter 2: Mass spectrometry of biomolecules Ionization by laser desorption Matrix assisted laser desorption ionization Time-of-flight mass spectrometry Mass spectrometric peptide mapping Protein identification by PMF
21 Analysis Protein of macromolecules identification why? 1.) Comparison of samples healthy control tumour tissue
22 Protein identification 2.) Identification of interacting proteins Protein binding to antigen Proteins isolated by immunoprecipation Antibody against known protein is added to mixture under which do not disrupt non-covalent bonds Antibody Antigen Proteins that interact with antigen are co-precipitated and usually subjected to SDS-PAGE (or even 2D-PAGE) Identification of regulatory proteins, functional clusters
23 Protein identification Identification by: 2D-electrophoresis (pi, M r ) Antibodies N-terminal sequencing
24 Protein identification Mass spectrometry for: Molecular mass of intact molecule Masses of enzymatically produced pieces peptide mass mapping Analysis of fragments of the pieces (MS / MS = Tandem MS) Identification / sequencing of proteins But: How to volatize a peptide or protein?
25 MALDI-TOF MS Laser desorbs (ablates) material from surface
26 MALDI A solution of a UV absorbing "matrix" is mixed with the sample. Evaporation of solvent leads to "co-crystallisation" of matrix and analyte. Matrix is ionized by firing a laser puls (2-3 ns). This also leads to soft desorption / ionisation of analyte molecules, even of large biomolecules such as proteins. hν
27 MALDI-TOF MS The mass of the ions is determined by a time-of-flight analyser (TOF MS). Laser pulse (t = 0) Matrix with sample (1 pmol) flight tube (1 m) Detector
28 MALDI-TOF MS Force constant
29 MALDI-TOF MS After generation, ions are accelerated in an electrical field. Thereby, the obtained velocity is proportional to the mass of the ions. Electric field Ionized sample molecules (amol) flight path (1 m) Detector
30 MALDI-TOF MS In the TOF: E p = U * q (Voltage and charge of particle E p = E k E k = ½ m * v 2 Force kinetic energy m/z = a b t 2
31 MALDI-TOF MS The time required to pass the drift tube is measured. Electric field flight path (1 m) Detector with watch (oscillator)
32 Low mass analysis by MALDI-TOF MS matrix ions MALDI is NOT a method for small molecules m / z
33 MALDI-analysis of a protein MH The major ion species has charge 1, even in the case of very large molecules M 2 M 3 2MH m / z
34 MALDI analysis of a peptide mixture Peptides obtained by trypsin digestion of a protein Each peak represents a distinct peptide mix? m / z
35 MALDI peptide mapping Preparation of the sample Excised spot is destained and dried Reduced and S-alkylated (iodoacetic acid, iodoacetamide, 4-vinylpyridin) see next page Digested with trypsin (or other protease, e.g. Endo-Glu-C ( V8 ), chymotrypsin..) Peptides are extracted and subject to analysis
36 MALDI analysis of a peptide mixture R 8 O N C C H R 27 N C C H N C C H CH2 S S CH2 O N C C R 10O N C C H H R 29 N C C Mercaptoethanol HS SH OH OH SH Dithioerythritol CH2 CH2 H O H H O H H O Reduction of disulfid bridges with 2-Mercaptoethanol, DTT or DTE Protein with -S-S- bridges between two Cys residues R R 8 O H O O S-Alkylierung / Carboxymethlyierung stabil... CH2 CH2 N C C H COO S H O... Reaction with Iodoacetic acid (or iodoacetamide) N C C H R 27 N C C H O N C C H CH2 SH SH CH2 N C C H H O HO OH SH OH SH Dithiothreitol (rac.) 10 N C C H H R 29 N C C H H O... unstable, reoxidizes...
37 1 MMGLLTNLRG SRTDGAQQDS LPVLAPGGNP KRKWSNLMPL VVALVVIAEI AFLGRLDMAK 61 NAAMVDSLAD FFYRSRAVVE GDDLGLGLVA SDRNSESYSC EEWLEREDAV TYSRDFSKEP 121 IFVSGADQEW KSCSVGCKFG FSGDRKPDAA FGLPQPSGTA SILRSMESAE YYAENNIAMA 181 RRRGYNIVMT TSLSSDVPVG YFSWAEYDMM APVQPKTEAA LAAAFISNCG ARNFRLQALE 241 ALEKSNIKID SYGGCHRNRD GRVNKVEALK HYKFSLAFEN SNEEDYVTEK FFQSLVAGTV 301 PVVVGAPNIQ DFAPSPGSIL HIKEIEDVES VAKTMRYLAE NPEAYNQSLR WKYEGPSDSF 361 KALVDMAAVH SSCRLCIHLA TVSREKEENN PSLKRRPCKC TRGPETVYHI YVRERGRFEM 421 ESIYLRSSNL TLNAVKAAVV LKFTSLNLVP VWKTERPEVI RGGSALKLYK IYPIGLTQRQ 481 ALYTFSFKGD ADFRSHLENN PYAKFEVIFV mix? m / z
38 Peptide Mass Fingerprinting with MALDI MS cleavage sites T1 T2 T3 T4 T5 T6 T7 T7 T5 T2 T3 T1 T4 T6 Peptides are of different length Peptides have different amino acid sequence (and composition) Amino acids differ in mass (except Ile = Leu) (difficult to discriminate: Lys and Gln; Met-SO and Phe)
39 Peptide Mass Fingerprinting with MALDI MS List of experimental peptide masses List of in silicopeptide masses of all proteins in databank #1 #2 #3 # etc. Search for best match (most peaks, most accurate) Problem: can only work with pure proteins
40 Peptide Mass Fingerprinting with MALDI MS Consider a decapeptide amino acids Question 1: how many sequences? Question 2: how many masses? (note on the side: very similar masses may not be discernible) Consequences? 6 if we measure 1 peptide from a protein? if we measure 4 peptides from a protein??
41 Peptide mass fingerprinting Risk of false identifications depending on: - Number of peptides - Size of protein -...?... (imagine: you live in an imperfect world!)
42 Role of measurement error in PMF mass error because of - calibration - reproducibility -peak shape error tolerance large: many false hits error tolerance small: few false hits calc. mass exp. mass matter of instrument and calibration (see later) Strategies: 1 - better PMF data 2 - totally different approach
43 MALDI analysis of a peptide mixture (in 1999!) experimental calculated mass of MH / average error (Da) residues sequence 2023,8 2023,1 0, FSLAFENSNEEDYVTEK 1927,6 1927,2 0, KPDAAFGLPQPSGTASILR 1624,2 1623,8 0, TEAALAAAFISNCGAR 1418,1 1417,6 0, ALVDMAAVHSSCR 1335,0 1334,5 0, GPETVYHIYVR 1 mmglltnlrg srtdgaqqds lpvlapggnp krkwsnlmpl vvalvviaei aflgrldmak 61 naamvdslad ffyrsravve gddlglglva sdrnsesysc eewleredav tysrdfskep 121 ifvsgadqew kscsvgckfg fsgdrkpdaa fglpqpsgta silrsmesae yyaenniama 181 rrrgynivmt tslssdvpvg yfswaeydmm apvqpkteaa laaafisncg arnfrlqale 241 aleksnikid syggchrnrd grvnkvealk hykfslafen sneedyvtek ffqslvagtv 301 pvvvgapniq dfapspgsil hikeiedves vaktmrylae npeaynqslr wkyegpsdsf 361 kalvdmaavh sscrlcihla tvsrekeenn pslkrrpckc trgpetvyhi yvrergrfem 421 esiylrssnl tlnavkaavv lkftslnlvp vwkterpevi rggsalklyk iypigltqrq 481 alytfsfkgd adfrshlenn pyakfevifv
44 MALDI analysis of a peptide mixture Peak masses are now entered into a Web-based program which compares your masses with in silico digests of proteins in data bank. You have to select: cleaving agent (trypsin, CNBr, others) mass tolerance possible modifications (S-acetamidomethylation) average mass or mono-isotopic mass protein features (mass, pi, species) 1. gi Mass: Total score: 62 Peptides matched: 5 (Y18529) Fuct c3 protein [Vigna radiata var. radiata]
45 MALDI analysis of a peptide mixture Why is identification rather poor? Only a few masses accuracy poor! Why so few peptides? Hydrophobic peptides not eluted smaller peptides (< 800 Da) poorly detectable larger peptides (> 3000 Da) poorly detectable some peaks come from trypsin or impurities some peptides arise from mis-cleavages others are "suppressed"
46 Peptide mass fingerprinting - PMF Idea: 1 protein in spot, 12 major peptides mass (m/z)
47 Peptide mass fingerprinting - PMF Idea: 1 protein in spot, 12 major peptides Reality I: - some peptides are modified (artefacts or real modifications) - some Lys/Arg sites are not cleaved (0-100 %) - many peptides do not show up (suppression etc.) Reality II: - peaks from trypsin or other background signals Reality III: - peaks from other protein Cys-containing peptide Cys-CAM / Cys-PAM Diff = 14 Da Met-containing peptide Met / MetOx Diff = 16 Da 1 missed cleavage mass (m/z)
48 Chapter 3: Resolution and isotope patterns Improving resolution in MALDI-TOF MS Resolution in mass spectroscopy Isotope patterns Exact mass
49 Improved resolution and accuracy in MALDI-TOF MS Performance is improved -by delaying the application of the acceleration field "delayed extraction
50 Improved resolution and accuracy in MALDI-TOF MS Resolution in "linear" MALDI-TOF instrument limited. Performance is improved -by redirecting the ions in a reflector (compensates for dispersity of kinetic energy) Modern instruments combine both abilities and achieve resolutions well above 10000, i.e. unit resolution for most peptides.
51 Resolution R = 500 R = 2000 unit resolution R in FWHM (full width at half maximum)
52 Carbon occurs as 13 C at appr. 1.1 % C 64 H 98 N 18 O 13 S m / z
53 Mass Abundance Diff Isotopes of the elements most relevant in biochemistry H C N O Na P S K
54 Isotope pattern of a peptide of mass ca monoisotopic mass average mass m/z
55 Isotope pattern observed at medium resolution Isotope pattern observed at ultrahigh resolution delta N S C O H , ,0 1185,5142 4,4 1185,5166 0,8 1185, ,3 1185,5214 0,6 1185,5235 0,9 1186,5113 0,1 1186,5130 4,5 1186,5176 2,5 1186,5200 0,5 1186,5215 3,5 1186, ,1 1186,5248 0,4 1186,5268 0,5 1187,5100 0,2 1187,5164 2,6 1187,5185 0,2 1187,5210 0,7 1187,5233 0,1 1187,5248 2,0 1187,5273 3,0 1187,5281 0,1 1187,5302 0,1 1188,5134 0,1 1188,5173 0,2 1188,5197 0,7 1188,5218 0,1 1188,5243 0,1 1188,5257 0,1
56 Carbon occurs as 12 C and to ca. 1.1 % as 13 C The origin of isotope patterns Val-Asn-Phe-Tyr-Ala-Trp-Lys C 47 H 62 N 10 O 10 Average mass: Monoisotopic mass: One molecule with 10 carbon atoms Many of them % m/z
57 Isotope patterns of larger peptides monoisotopic mass m/z m/z
58 Isotope patterns of larger peptides An allergen from apple
59 Isotope patterns of a large protein
60 Expressions of mass Monoisotopic mass (exact mass) only up to ca Da Exact mass of a given compound is the sum of the exact masses of only the most abundant isotopes for each element present. For small molecules, elemental composition can be deduced from the exact mass. Average mass Weighted average of all isotope peaks Coincides at higher mass with peak top
61 Expressions of mass Nominal mass: integer mass value for the most abundant isotope (H = 1, C = 12 etc.) Monoisotopic mass: exact mass value for the most abundant isotope (H = , C = etc.) Average mass: chemical (average) atomic mass value (H = , C = etc.) Example: CYIQNCPLG vs Da
62 Ions are detected as peaks and not as lines I.e.with a statistical error of mass value monoisotopic mass m/z
63 Resolution in mass spectrometry monoisotopic mass monoisotopic mass monoisotopic mass monoisotopic mass peak top average mass average mass m/z Theoretical R = 5000 R = 1500 R = 500 m/z Resolution as FWHM = full width at half maximum
64 Resolution in mass spectrometry
65 Resolution is determined by instrumentation and proper use thereof Linear MALDI-TOF R 500 Resolution enhances by - delayed extraction - reflectron Reflectron DE-MALDI-TOF R = around Highest resolution at minimum laser energy required to just effect analyte desorption. Consequence: bad signal : noise ratio Solution: Summing of many individual spectra
66 MALDI-TOF MS Significance of resolution MALDI-Q-TOF MS von Wespengift Hyase
67 Chapter 4: Practical aspects of MALDI-TOF MS Matrices Preparation of sample spots Acquiring data Identification of the type of ion
68 MALDI matrix substances α-cyano-4-hydroxycinnamic acid for peptides ACH, HCCA (MH = 190,0504 / 190,18) 1 % solution in 70 % acetonitrile (optional: 0.1 % TFA) 2,5-dihydroxybenzoic acid general purpose, carbohydrates DHB, gentisic acid (MH = 155,0344 / 155,13) 2% in 30 % acetontrile (and other recipes) sinapinic acid, ferulic acid protein 1 % solution in 70 % acetonitrile (optional: 0.1 % TFA) and many others for more special purposes e.g. for glycans: 1-hydroxyisoquinoline (HIC), D-arabinosazone, 6-aza-2-thiothymine (ATT), 2,4,6-trihydroxy-acetophenone (THAP), 3-aminoquinoline etc.
69 For peptides: MALDI sample preparation 1 µl containing 0.5 to 10 pmol peptide is spotted on the target. Method 1: Sample is dried; 1 µl ACH is added and allowed to dry. Method 2: ACH is added to sample drop and mixed by pipetting. Method 3: Sample and matrix are premixed in vial. For carbohydrates: Option: acidify matrix with TFA or formic acid 1 µl containing 2-50 pmol oligosaccharide is dried on the target 1 µl DHB is added and the drop is dried immediately in vacuo. For proteins: (or re-cristrallysed by addition of 1 µl ethanol) Equal volumes of sample and matrix are premixed. 1 µl is spotted on target and air dried. Option: acidify matrix with TFA or formic acid
70 Samples for MALDI MS MALDI is relatively tolerant towards salts and other impurities if too much: no cristalline sample surface Upper limits for selected additives: NaCl 50 mm Phosphates 10 mm Tris 50 mm Urea 1 M Glycerol 1 % SDS 0,01 % Tween, Triton X-100, NP-40 0,1 % n-octyl-ß-glucoside 1 %
71 Desalting samples for MALDI MS Desalting of peptides and proteins may be achieved by means of reversed phase extraction using small volumes of RPmaterial in mini-columns or premade tips (Zip-tips). RP18 for peptides RP4 for proteins RP-material must be prewetted with high AcCN solvent, then equilibrated with aqueous solvent (water TFA or formic acid). Peptides are now bound, salts are eluted with (acidified) water. Peptides are eluted with AcCN/water mixture.
72 Desalting samples for MALDI MS Desalting of sugars can be done with ion exchange resins such as Dowes 50W X8 or Dowex 1 X8. Alternative: matrices which bind the sugar: porous graphitized carbon (PGC) sugar adsorbed from aequous solvent eluted with % acetonitrile hydrophilic adsorbents (HILIC) (Diol-, Amino-, Amide-, Cyano-matrices) binding in 96 % AcCN elution with 50 % AcCN
73 Acquiring a MALDI-TOF spectrum laser power desorption threshold (resolution, accuracy) position sweet spot, not always uniform distribution of sample accumulation of data. signal to noise ratio
74 Types of (pseudo)-molecular ions Peptides: [M H] Proteins: [M H], [M 2H], [2M H] etc. Sugars: [M Me], (usually Na, some K) Acidic glycans: [M x Me], (carboxylgroups as salt; -COONa) Nucleic acids glycans: [M - H] -, [M - 2H Na] -, (carboxylgroups as salt; -COONa)
75 Determining the ion type Example: peptide, M = [MH] H... 1 Na K [MNa] [MK]
76 Determining the ion type Example: glycopeptide H... 1 Na K [MNa] [MH] [MK]
77 Determining the ion type Example: oligosaccharide, M = high resolution [MNa] [MK]
78 Determining the ion type Example: oligosaccharide mixture high resolution K-peaks unusually strong [MK] 100 [MNa] [MK] % [MNa] [MNa] [MK] m/z
79 Chapter 5: Fragment ions in MALDI-TOF MS Post-source decay Fragmentation of peptides peptide sequencing by MS
80 Metastable ions in MALDI-TOF MS MALDI is a very soft ionisation process but - excess energy may be deposited on ion - cannot be dissipated to other molecules (vaccum) - "slow" fragmentation ad fragile bonds will occur somewhere during flight of ion hence post source decay M ---> F 1, F 2, F 3... PSD fragment ions have same velocity as precursor but smaller mass. In linear TOF not discriminated
81 MALDI-TOF MS In "normal" reflectron operation, only the intact (pseudo)-molecular ions are observed. Hence, often larger molecules are less efficiently detected in reflectron mode. The reflectron enables to analyse the fragment ions from a particular precursor mass (time window). acc.-field Deflector (mass selector)
82 MALDI-TOF MS MALDI-TOF MS spectrum of a peptide mixture m/z
83 Peptidsequenzierung mittels PSD-MALDI or Tandem MS (= MS/MS) Spaltstellen T1 T2 T3 T4 T5 T6 T7 Mischung von tryptischen Peptiden T7 T3 T1 T5 T4 T2 T6 Auswahl eines Ions im MS1 T4 T4 T4 T4 T4 Fragmentierung, Bestimmung derfragmente im MS2 Breaking points are the peptide linkages between amino acids
84 MALDI-TOF MS MALDI-TOF PSD spectrum of a selected peptide highly simplified precursor S WL T P N G D M T y 1 y 2 y 4 y 5 y 6 y 7 y 8 y 9 y 10 fragment ions
85 MALDI-TOF MS Major fragments derived from a peptide (protonated) y 3 y 2 y 1 H H H R 1 R 2 R 3 R 4 H N C C N C C N C C N C COOH 2 H O H H O H H O H H b 1 b 2 b 3 immonium ions from single amino acids H 2 N = CHR n
86 The y-series (y ) precursor MALDI-TOF MS R 1 R 2 R 3 NH x H 2 N C C N C C N C C N C COOH H O H H O H H O H H Lys or Arg R 2 R 3 R 4 y 3 H 2 N C C N C C N C COOH O O H H H H H R 3 R 4 y 2 H O H H H 2 N C C N C COOH
87 MALDI-TOF MS The y-series precursor T M D G N P T L W S G R Thr-Met-Asp-Gly-Asn-Pro-Thr-Leu-Trp-Ser-Gly-Arg y 11 = Met-Asp-Gly-Asn-Pro-Thr-Leu-Trp-Ser-Gly-Arg y 10 = Asp-Gly-Asn-Pro-Thr-Leu-Trp-Ser-Gly-Arg y 9 = y 8 = y 7 = y 6 = Gly-Asn-Pro-Thr-Leu-Trp-Ser-Gly-Arg Asn-Pro-Thr-Leu-Trp-Ser-Gly-Arg Pro-Thr-Leu-Trp-Ser-Gly-Arg Thr-Leu-Trp-Ser-Gly-Arg y 5 = Leu-Trp-Ser-Gly-Arg
88 MALDI-TOF MS Masses of immonium and other small ions m/z amino acid present 70, 112 arginine 70 (without 112) proline 72 valine 86 leucine / isoleucine 110 histidine 136 tyrosine 147 acrylamide-modified cysteine 159 tryptophane 216 phosphotyrosine
89 MALDI-TOF MS Problematic amino acids proline signal of fragment often weak cysteine forms adducts with acrylamide before carboxymethylation leucine / isoleucine have identical mass glutamine / lysine have almost identical mass ( v ) methionine can be oxidized to methionine-sulfoxide ( = and methionine-sulfonic acid (32) Phe = )
90 MALDI-TOF MS Sequence information from MALDI-TOF PSD spectra Post source decay: modern terms : LID laser induced decay LIFT... laser induced xxxxx Interpretation of spectra difficult (not only y-fragments) Sequence information from old-style MALDI-TOF PSD spectra not generally satisfactory. Improved by chemical derivatisation to enhance y-series and allow unambiguous interpretation (e.g. negatively charged group on N-terminus).
91 MALDI-TOF MS Sequence information from MALDI-TOF PSD spectra Wonderful idea -- poor technique - ESI-MSMS - ESI-iontrap-MS n - MALDI-TOF/TOF (with LIFT technology or alike)
92 MALDI TOF-TOF MS capable of CID-MSMS Principle: 2 kick-and-fly zones in one Instrument Allows to use PSD as well as CID (collision induced decay)
93 MALDI TOF-TOF MS Bruker Autoflex 1000 Hz Resolution of over FWHM TOF/TOF with either LID (laser induced decay) or high-energy CID whole proteins peptide & peptide ID sugars etc.
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