The Nobel Prize in Chemistry 2002

Transcription

1 The Nobel Prize in Chemistry 2002 With mass spectrometry we can now quickly identify a substance in a sample by accurately determining its molecular mass. is a very widely used method for small and medium-sized molecules. The trick was to get the proteins to fly, or as John Fenn himself said, to give "wings to molecular elephants". 1 From: 2 I hope he weighs above 120 lb so I do not have to fight him. I want 50 mg of NaCl. 3 4 Living systems contain many different units of varying size with well defined masses Many of these masses are (more or less) unique. Atoms and molecules can be identified by their mass. The structure of a molecule can be investigated by weighing the mass of its fragments. Elem. Isotope Mass H 1 H H C 12 C C N 14 N N O 16 O O O P 31 P S 32 S S S S Cl 35 Cl Cl K 39 K K K Valine C 5 H 11 ON = Alanine C 3 H 7 ON =

2 Residue Mass Gly G Ala A Ser S Pro P Val V Thr T Cys C Ile I Leu L Asn N Asp D Gln Q Lys K Glu E Met M His H Phe F Arg R Tyr Y Trp W H 2O Asp-Gly-Glu-Ala = Asp-Gly-Gln-Ala = Asp-Gly-Lys-Ala = Modificeret tabel fra Analysis of recombinantly expressed protein and of synthetic peptides Protein identification and sequencing Identification and localization of posttranlational modifications Identification and stochiometry of protein complexes Identification of protein domains Disease -- protein expression 7 8 Measure low (picomolar) concentrations with resolution (e.g. 4Da for a MW of 40 kda molecule) Fragment the ions, perform chemical reactions on them, etc. Run several consecutive MS experiments Interface with other separation techniques e.g. GC, LC electrophoresis 19 publications this year in Science, Nature, and Cell 9 10 An instrument that produces ions and separates them in the gas phase according to their mass-to-charge ratio () Sample Vacuum system Sample introduction device Ionization source Mass analyzer Ion detector Data analysis system Figure from

3 Charge: Since it is difficult to measure the gravitational forces on molecular size objects, we will look at: the laws of motion electric fields magnetic fields e = C Mass: 60 W 220 V (1 million) 3 e/s 1 Da = kg 1 g / (1 million) F v m V acc /2 -V acc /2 V acc /2 -V acc /2 q F m F q m d d

4 L v q m m z - higher lower Fast detection (100 spectra / s) Spectrum collected in one shot Unlimited Needs pulsed source Da 1 20 kv - m 2 2m 2 m 2m t 1 m 0 t / 2 t t 2 v = m/s t / 2 t t 2 t = 10 µs 0 m/2 m 2m bovine insulin (5.7 kda) [MH] [MmatrixH] [2MH] [3MH] [M2H] 2 Figure from

5 v B q m F Lorenz v m F r v q m F r B B B r r m z - B (or V) is scanned higher or lower B lower or higher B 0 m 2 m B / 2 B 2m B 2 B (~30 cm) B/ 2 B B 2 Classical reference instrument Best quantification High resolution (slit width) Large and expensive Scanning (jumped) < m/2 m 2m Modificeret Figure from

6 mass mass R=720 bovine proinsulin C 381 H 586 N 107 O 114 S 6 monoisotopic mass: Da average mass: Da higher resolution Elem. Isotope Mass Abundance (%) H 1 H H C 12 C C N 14 N N O 16 O O O P 31 P S 32 S S S S Cl 35 Cl Cl R=9500 K 39 K K K Ions can be stored in the trap U/V is scanned Can be used for chemical reactions Ejects ions of increasing mass Figure from Small and inexpensive Suitable for MS-MS Limited resolution Peak height function of mass Scanning (jumped) < Figure from Sheehan: Physical Biochemistry. Similar to quadrupole mass filter but poorer for analysis <

7 Figure from theses2001/perretc/these_body.html Molecules are trapped in the field of a superconducting magnet Detection and excitation using RF coils like in NMR Highest resolution (10 6 ) Non destructive detection Advanced manipulation of ions Well suited for pulsed sources Very large and expensive TOF Fast detection (100 spectra/s) Spectrum collected in one shot Unlimited Needs pulsed source Quadrupoles Small and inexpensive Suitable for MS-MS Scanning (jumped) Trap can be used for storage and reactions Limited resolution (unit resolution 0.3 units) Peak height function of mass Magnetic sector Classical reference instrument Best quantification High resolution (slit width) Large and expensive Scanning (jumped) Ion cyclotron resonance Highest resolution (10 6 ) Non destructive detection Advanced manipulation of ions Well suited for pulsed sources Very large and expensive An instrument that produces ions and separates them in the gas phase according to their mass-to-charge ratio () Sample Vacuum system Sample introduction device Ionization source Mass analyzer Ion detector Data analysis system Figure from Heated Filament Beam of electrons 41 Figure from Sheehan: Physical Biochemistry. 42 7

8 molecules stripped of e - molecular ion (unstable) Only ions with the right sign of charge visible fragment ions (structure dependent) multiply charged ions Figure from Sheehan: Physical Biochemistry. Figure from Sheehan: Physical Biochemistry. Far too violent for macromolecules for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules High energy Xe, Ar, Ce 3 Liquid, e.g. glycerol (~90%) analyte ionized by matrix or T-increase singly charged ions dominate possible fragmentation of labile bonds peptides and oligosaccharides < 10 kda varying ionization efficiency Koichi Tanaka, Japan John B. Fenn, USA Figure from Koichi Tanaka Analyte ionized by matrix Singly charged ions dominate Laser UV λ 10 Hz 10 khz bovine insulin (5.7 kda) in sinapinic acid matrix [MH] [Mmatrix] Matrix molecule [M2H] 2 sinapinic acid 225 Da Modified figure from [2MH] [3MH] solid solution w/ 99.99% matrix UV λ

9 Electrospray ionization (ESI) Spray John Fenn Heated N2 cone voltage 4 kv _ Coulumbic explosion electron pump kv Evaporation Very gentle Sensitive to impurities Multiple charges ~1/kDa (Depending on folding etc.) Figures from Sheehan: Physical Biochemistry. Electrospray ionization (ESI) ESI spectrum horse myoglobin ( Da) deconvolution 52 Figure from Deconvolution 53 Nanospray ionisation for two consecutive peaks (i and i1) with charges zi and zi1= zi1: 1 10 µm V = 1-2 kv 1 µm 10 nl / min When the charges are known the molecular mass can be calculated with high accuracy: Figure from spectra / s Figure from Miranker, PNAS 97: (2000)

10 positive ion mode for proteins and negative ion mode for saccharides and oligonucleotides sample treatment MW limit pulsed / continuous EI FAB MALDI ESI nano spray harsh quite gentle gentle very gentle very gentle hundreds of Da ten kda hundreds of kda hundreds of kda both both pulsed both both hundreds of kda LC/MS gas phase yes possible yes too slow Charges mostly 1 mostly 1 mostly 1 ~1 per kda ~1 per kda Low MW background no yes yes no no Impurities very sensitive very sensitive Detection limit mol mol mol mol Perkin-Elmer omniflex $ 99,500 TOF needs a pulsed source. The ions with unit charge produced by MALDI demand detection of high mass compounds by a mass analyzer with a high upper limit. Preferred analyzer TOF quadrupole quadrupole Finnigan Deca The multiply charged ions from ESI permit detection of high mass compounds by most mass analyzers ( typically less than 2000 to 3000). Controlled buffer conditions (e.g. uniform oxidation state) Some ionization methods (e.g. FAB & MALDI) require special matrix molecules No salt (normally < 1 mm) No interference Tolerable (< 50 mm) Avoid TFA HEPES glycerole formic acid MOPS sodium azide β-mercaptoethanol Tris DMSO DTT NH 4 Ac SDS volatile organic solvents HCl NH 4 OH octyl glucoside phosphate NaCl urea acetic acid guanidine

11 To avoid colliding with other particles p < 10-4 Pa (10-9 atm) Exception: p = 0.1 Pa (10-6 atm) for quadrupole ion trap Chromatography Interface Detector Source MS1 Detector Additional information Simpler No impurities No supression effects Minimal loss Not all column types Interface Figure from quadrupole ion trap ion cyclotron backbone: sidechain: inert gas e.g. Ar, He Figure from Source Scan or selection MS1 Collision Detector Cell MS2 Detector ELECTROSPRAY NEEDLE All ions My ion My ion plus its fragment ions Ion separation Collision induced decomposition MS1 MS2 Figure from MS-MS in space or in time selection both in MS1 and MS

12 MS1 MS2 product ion scan parent ion scan selection in MS1 sensitivity in MS2 68 Figure from MStut/mstutorial.htm 69 MS N, N<12 usually 3 for proteins MS N, N<12 usually 3 for proteins MS N, N<12 usually 3 for proteins MS N, N<12 usually 3 for proteins

13 MS N, N<12 usually 3 for proteins MS N, N<12 usually 3 for proteins MS 3 on the triply-charged ion from ACTH, a 22 amino acid peptide. A fragment ion at 505 was chosen for further fragmentation Analysis of recombinantly expressed protein and of synthetic peptides Protein identification and sequencing Identification and localization of posttranlational modifications Identification and stochiometry of protein complexes Identification of protein domains Disease -- protein expression

14 Amino acid fragment weights Ala A Ser S Pro P Val V Thr T Cys C Ile I Leu L Asn N Asp D Gln Q Lys K Glu E Met M His H Phe F Arg R Tyr Y Trp W Water Modificeret tabel fra bovine insulin (5.7 kda) 80 [3MH] Gly G Control of recombinant protein [2MH] Average mass [MH] Monoisotopic mass [M2H]2 Amino Acid Peptide synthesis 81 Analysis of synthetic peptides Attachment oxidation of Met8 Deblocking MH Cleavage tert-butyl Glu7Δ O-trifluoroacetyl Figure from Sheehan: Physical Biochemistry. 82 Proteases 83 Proteolytic digestion mass N C Subtilisin Thermolysin Chymotrypsin Endoprot. Glu-C Endoprot. Lys-C Trypsin preferentially neutral or acidic = Ala, Leu, Met, Val, Ile, Phe = Ala, Leu, Met, Phe, Trp, Tyr = Glu, Asp = Lys = Lys, Arg Endoprot. Glu-C Trypsin = Glu, Asp = Lys, Arg time N C Carboxypeptidase N C

15 trypsin R^, K^ K ^ TYFPHFDLSHGSAR ^ VK ^ ( = 1635) K ^ TYFPHFDLSHGSAQVK ^ ( = 1834) endoproteinase LysC K^ K ^ TYFPHFDLSHGSARVK ^ ( = 1862) Figure from Sheehan: Physical Biochemistry. Figure from Sheehan: Physical Biochemistry CID or proteolysis Carboxypeptidase MS MALDI-TOF LC-MS MS-MS LC-MS-MS N C Δm = 113 Ile/Leu Δm = 87 Ser Δm = 128 Gln/Lys LC-MS / MALDI-TOF MW Protein sample (e.g. from 2-D gels) Digest to peptides (preferably 2.5 kda or less) Make an initial MS showing MW of all components in the digest (peptide map) This may be enough for a database search and identification Fragment peptides along the amino acid backbone in tandem mass spectrometry Some peptides generate enough info for full sequence, others only generate partial sequences of 4-5 amino acids Often this tag sequence is sufficient for database identification pi

16 Database search Posttranslational modifications Ragged ends: Aminopeptidases D DS DSG DSGQ Carboxypeptidases Glycosylation Phosphorylation Acylation Disulphide formation 92 Posttranslational modifications Internal 93 Modification, adduct, etc. 30 Homoserine formed from Met by CNBr treatment 18 Dehydration ( H2O) 18 S-γ-glutamyl (crosslinked to cysteine) 17 Pyroglutamic acid formed from Gln 17 N-(β-aspartyl)-lysine (crosslink) 2 1 Figures from Sheehan: Physical Biochemistry. 95 Disulphide formation Disulphide bond formation (cystine) Deamidation from Asp and Glu to Asn and Gln 14 Methylation (N-terminus, Nε of Lys, O of Ser, Thr) 16 Oxidation of Met (to sulphoxide) 22 Na 32 Oxidation of Met (to sulphone) 38 K 41 Carbamylation 42 Acetylation (N-terminus, Nε of Lys, O of Ser) 43 N-trimethylation (of Lys) 44 Na2 58 Carboxymethylation (on cystine) 60 Na K 71 Acrylamide or acrylamide adduct 80 Phosphorylation (O of Ser, The, Tyr) 80 Sulphation (O of Tyr) 119 Mass changes Δm Cysteinylation 96 Identification of disulphide bridges non-reducing proteolysis MS reducing Figures from Sheehan: Physical Biochemistry

17 protease Limited proteolysis ph 5.2 ph 3.2 ph 2.6 Total proteolysis Electrospray mass spectra of bovine cytochrome c (12.5 kda). Higher charges (lower ) at lower ph are due to denaturation and exposure of more basic groups to the solvent. LC-MS / MALDI-TOF Billede fra calbindin binds 2 calcium ions cooperatively cone voltage Ca2 Ca 2 Ca 2 ESI or nanospray ionization Chazin & Veenstra, Rapid Commun Mass Spectrom 13: (1999) Figure from Miranker, PNAS 97: (2000) β α α β β β 2 α 4 β β β α α β β Fändich et al., PNAS 97: (2000)

18 Proteome of the human cornea Protocol 1 Proteins were extracted in urea or 100 mm NaCl Separated by 2D gel electrophoresis Proteins identified by MALDI-TOF MS Materials 16 normal human donor corneas Age years Containing all cell-layers Lyophilized and homogenized in N2 Fine corneal powder Karring et al., Molecular & Cellular Proteomics 4:1406 (2005) 106 Protocol 2 Karring et al., Molecular & Cellular Proteomics 4:1406 (2005) 107 Protocol 3 Proteins were extracted in SDS sample buffer and separated by SDS-PAGE The gel lane was sliced in ~2-mm strips and digested with trypsin The extracted peptides were identified by LC-MS/MS The corneal powder was solubilizedusing CNBr and trypsin The peptides were separated by off-line strong cation exchange and identified using LC-MS/MS Karring et al., Molecular & Cellular Proteomics 4:1406 (2005) 108 Proteome of the human cornea Karring et al., Molecular & Cellular Proteomics 4:1406 (2005) 109 Desorption electrospray ionization (DESI) Identified proteins: Urea, 2D-gels, MALDI-TOF MS: 67(165 spots) CNBr-trypsin, SCX, LC-MS/MS: 31 SDS-PAGE slices, LC-MS/MS: 103 Total number of different proteins:141 No sample preparation! Karring et al., Molecular & Cellular Proteomics 4:1406 (2005) 110 From: Cooks et al., Science 311:1566 (2006)

19 µ The product ion MS/MS spectrum identifies one of the minor components, with = 214, as aspartyl-4-phosphate. TNT, trinitrotoluene; RDX, hexahydrotrinitro-1,3,5-triazine; HMX, octahydro-mx H1,3,5,7-tetranitro-1,3,5,7-tetrazocine; PETN, pentaerythritol tetranitrate; TATP, triacetonetriperoxide From: Cooks et al., Science 311:1566 (2006) 112 From: Cooks et al., Science 311:1566 (2006) 113 Analysis of recombinantly expressed protein and of synthetic peptides Protein identification and sequencing Identification and localization of posttranlational modifications Identification and stochiometry of protein complexes Identification of protein domains Disease -- protein expression An instrument that produces ions and separates them in the gas phase according to their mass-to-charge ratio () Sample Vacuum system Sample introduction device Ionization source Mass analyzer Ion detector Data analysis system Figure from