Mass Spectrometry and Proteomics - Lecture 1 - Matthias Trost Newcastle University

Transcription

1 Mass Spectrometry and Proteomics - Lecture 1 - Matthias Trost Newcastle University matthias.trost@ncl.ac.uk

2 Content Lectures 1-3 The basics of mass measurement Ionisation techniques Mass analysers Detectors Tandem mass spectrometry Fragmentation techniques Peptide fragmentation Hybrid instruments Lecture 1 Lecture 2 Lecture 3 2

3 Content Lectures 4-6 What is proteomics? Sample Preparation Experimental Design Quantification techniques Search engines, Databases, FDR Data analysis & Data inspection Fractionation techniques Phosphoproteomics and other PTMs Proteomics experiments Lecture 4 Lecture 5 Lecture 6 3

4 Lecture 1 Basics Components of a mass spectrometer Isotopes and isotopic profiles Resolution Accuracy vs. Precision Ionisation techniques Electrospray Ionisation Matrix-assisted Laser Desorption/Ionisation 4

5 The basics of mass measurements The zeroth law of mass spectrometry: Never ever say mass spectroscopy! Spectroscopy involves the measurement of electromagnetic waves and we look at particles. 5

6 History of mass spectrometry 1886 E. Goldstein discovers anode rays in a gas discharge tube J.J. Thomson discovers the electron and determines its m/z (Nobel Prize in 1906) 1912 J.J. Thomson constructs the first mass spectrometer and sees spectra of O 2, N 2, CO, CO 2 and COCl 2. He observes negative ions, multiply charged ions and identifies isotopes of 20 Ne and 22 Ne A.J. Demster develops the electron impact ion source and constructs the first mass spectrometer that allows focusing of ions in direction F.W. Aston constructs the first mass spectrometer that allows focusing of ions by velocity (Nobel Prize 1922) E. O. Lawrence invents the cyclotron (Nobel Prize in 1939) J. Mattauch and R. Herzog develop the first mass spectrometer that allows focusing of ions in direction and momentum with an electrostatic sector and a magnetic sector W.R. Smythe, L.H. Rumbaug and S.S. West perform the first preparative separation of isotopes A.O. Nier et al isolate the isotope 235 U. 6

7 History of mass spectrometry 1942 First commercial sector mass spectrometer by CEC A.E. Cameron et D.F. Eggers elaborate the plan for a Time-of-Flight mass spectrometer after a principle proposed by W. Stephens in Quasi-equilibrium theorie (QET) and Rice Ramsperger Kassel Marcus (RRKM) theory explain the molecular fragmentation of ions. Marcus receives Nobel Prize in W. Paul and H.S. Steinwedel describe the first ion trap mass spectrometer. W. Paul, H.S Reinhard and U. von Zahn publish the first quadrupol mass spectrometer. Paul and Dehmelt ( Penning trap ) receive Nobel Prize in J. Beynon show the first identification of the empirical formula through measurement of exact mass. First GC-MS by F.W. McLafferty and R.S. Gohlke M.S.B. Munson et F.H. Field introduce the chemical ionisation. K. Biemann et al determine first peptide sequence by mass spectrometry F.W. McLafferty and K.R. Jennings introduce collision-induced dissociation (CID) Finnigan commercialises the first quadrupol mass spectrometer. 7

8 History of mass spectrometry 1972 V.I. Karataev, B.A. Mamyrim et D.V. Smikk introduce the first Time-of-Flight mass spectrometer with reflectron M.D. Comisarov and A.G. Marshall apply the Fourier transformation to analyse ion cyclotron resonance mass spectra. P.J. Arpino, M.A. Bladwin and F.W. McLafferty present the first mass spectrometer coupled to a liquid chromatography system R.A. Yost and C.G. Enke construct the first triple quadrupol mass spectrometer M. Barber, R.S. Bordoli, R.D. Sedgwick and A.H. Tyler describe the atom bombardment ion source and publish the first spectrum of Insulin in Sciex and Finnigan commercialise the first triple quadrupol mass spectrometer 1987 M. Karas and F. Hillenkamp develop matrix-assisted laser desorption/ionisation (MALDI), K. Tanaka laser desorption. Tanaka receives Nobel Prize in J. Fenn develops electrospray ionisation after a concept proposed by M.Dole in Fenn receives Nobel Prize in A.A. Makarov presents a new type of mass analyser the Orbitrap D.F. Hunt lab develops electron-transfer dissociation (ETD) mass spectrometry. 8

9 The basics of mass measurements Sample introduction Ionisation Mass analyser Detector Data Acquisition and Analysis Chromatography (GC, HPLC) Direct injection Capillary electrophoresis ESI MALDI FAB CI FD/FI II Quadrupol Ion trap TOF FT-ICR Sectors (B, E) Orbitrap Electron multiplier Microchannel plate Ion-to-photon detectors FT-ICR Orbitrap Computer Vacuum 9

10 The basics of mass measurements: vacuum Vacuum technology Pressure (mbar) Pressure (mtorr) Vacuum torr-750 mtorr Primary Vacuum Intermediate Vacuum * 10-5 High Vacuum <10-7 <7.5 * 10-5 Ultra-high vacuum Rotation pumps (backing/roughing pumps): 4-16 m 3 /h for the primary vacuum necessary for turbomolecular pumps (turbo pumps). Ultra-high vacuum almost entirely achieved by turbo pumps ( L/sec) (20-90,000 rpm!, up to several thousand km/h!). Less used are diffusion pumps ( L/sec) and cryo-pumps. 10

11 The basics of mass measurements: vacuum Why is a vacuum necessary? The mean free path,, is the average distance traveled by an ion before it collides with an air molecule, and is given by: = 1/N where N is the gas number density, and is the collision cross section between the ion and the molecule (typically ~50 Å 2 for a small peptide ion). Using a collision cross section of 50 Å 2, the following table may be constructed: 11

12 The basics of mass measurements Positive-ion mode: the molecule with an additional proton Negative-ion mode: the molecule with a proton less. The calculated mass can be obtained from the empirical formula: C 11 H 10 N 3 Cl In the literature, the molecular mass can be shown as: Normalised on the most abundant peak Average mass: Da Nominal mass: 219 u Monoisotopic mass: u m/z: Th 12

13 Isotopes The basics of mass measurements: isotopes A molecule is defined by its empiric formula Each atom has a natural isotopic ratio due to difference in the number of neutrons. E.g. carbon and chlorine: 12 C: u <-> 13 C: u 35 Cl: u <-> 37 Cl: u 1u=1 Da=1/12 of 12 C ~ mass of the H-atom ( u) Each isotope has a natural abundance: E.g. 12 C: 100% <-> 13 C: 1.08% 13

14 The basics of mass measurements: isotopes Symbol #atomic Nominal mass Isotopic Composition Isotopic mass Average Mass H Na P C N O S Cl Br

15 Mass Defect The mass of an atom is less than the sum of the individual parts (protons, neutrons and electrons). This difference is called mass defect. The mass defect originates from the binding energy of protons and neutrons in the nucleus. The energy can be calculated by E=mc

16 The basics of mass measurements: isotopes Isotope pattern is dependent on the composition and the number of atoms. In larger biomolecules, the 13 C-peak becomes the main peak. ~75 C-Atoms ~100 C-Atoms ~125 C-Atoms 16

17 The distances between isotopic peaks reveal charge state mix of 6 proteins LCT prot_mix_0724a 651 (10.856) Sm (SG, 2x6.00); Cm (648:651) protein_modeling TOF MS ES+ 783 % 1.00 mix of 6 proteins LCT rot_mix_0724a 350 (5.837) Sm (SG, 2x6.00); Cm (343:374) protein_modeling TOF MS ES+ 1.86e m/z % mix of 6 proteins prot_mix_0724a 655 (10.923) Sm (SG, 2x6.00); Cm (645:675) LCT protein_modeling TOF MS ES+ 454 m/z % m/z

18 Resolution The basics of mass measurements: resolution 18

19 The basics of mass measurements: resolution Resolution How does the isotopic pattern vary with resolution for a peptide of 2000 Da? 19

20 The basics of mass measurements: resolution Resolution Impact on the identification of an ion species: C 20 H + 9 R=1000 R>10000 C 19 H 7 N + C 13 H 19 N 3 O 2 + C C 20 H + 19 H 7 N + 9 C 13 H 19 N 3 O Typical resolution of mass spectrometers: Quadrupol, Ion trap: <10,000 Orbitrap: up to 500,000-1,000,000 Time-of-Flight: 10-30,000 FT-ICR >1,000,000 20

21 The basics of mass measurements: Accuracy and Precision 21

22 Ionisation techniques Electrospray ionisation (ESI) Matrix-assisted laser desorption/ionisation (MALDI) Not covered: Electron Ionisation (EI), Chemical Ionisation (CI), Fast-Atom Bombardment (FAB) 22

23 Electrospray 23

24 Electrospray 24

25 Electrospray Ionisation (ESI) Ionisation of molecules from solution Soft ionisation technique Ease of coupling with separation techniques such as nano-lc Production of multiply charged ions ( MS/MS) 25

26 Electrospray ESI of large peptides and proteins: Production of multiply charged species: [M+zH] z+ Space between two ions corresponds to the difference of one charge. 26

27 How to determine the molecular mass of a protein from an ESI-MS spectrum Observed ions have composition [M+nH] n+ For the charge states of m/z 1 (higher value) and m/z 2 (lower value) we have n 2 =n m/z n must be an integer m n = m/z 1 = (H=mass of proton) Its neighbouring peak to the left: m n+1 =m/z 2 = = (with M being the mass of the protein) Solving both equations for n and M: / n 1 = / / M = n 1 (m/z 1 H) e.g. m/z 2 = and m/z 1 = n 1 = 17, and M = Da 27

28 Deconvolution of ESI mass spectra Charge states Deconvoluted spectrum M = n(m n H) Mass (Da) 28

29 Nobel Prize in Chemistry ) For the development of Electrospray Ionisation For the development of Desorption Ionisation Michael Karas Franz Hillenkamp 29

30 Matrix-assisted Laser Desorption/Ionisation (MALDI) Analyte is co-deposited with Matrix. Laser excites matrix which transfers energy to analyte. Produces predominately singly charged species [M+H] +. Typically used for large biomolecules / polymers. MALDI is a high mass/pulsed source so usually combined with TOF. Less sensitive to contaminants such as salts and detergents Sensitivity at attomole level. High throughput analysis (up to ~3000 samples/day) Samples can be re-analysed. 30

31 MALDI sample preparation Sample/matrix mix (1:10,000 molar excess) in volatile solvent. Requires only femtomoles of analyte. Drying Sample target 80x magnification of dried sample/matrix drop on target 31

32 Matrix-assisted Laser Desorption/Ionisation (MALDI) 1: peptides, 2: proteins, 3: oligosaccherides, 4: Nucleic acids, 5: polymers 32

33 Matrix-assisted Laser Desorption/Ionisation (MALDI) 33

34 MALDI matrix Absorbs photon energy and transfers it to analyte. Minimises aggregation between analyte molecules. Matrix must Absorb strongly at Laser wavelength. Have low sublimation temperature. Have good mixing and solvent compatibility with analyte. Have ability to participate in photochemical reaction. 34

35 Matrices and analytes: desired photochemical characteristics Absorbance Laser matrix analyte 200 Wavelength (nm) 500 Common lasers; N 2 (337 nm), ArF excimer (193), Nd-YAG frequency tripled (355 nm) and quadrupled (266 nm) 35

36 Applications: Mass determination of intact proteins Intens. [a.u.] x104 [M+2H] DHAP_forhighermasses_plusMax 0:G22 MS Raw [M+3H] [M+H] m/z MALDI-TOF spectrum of a single protein 36

37 Applications: Molecular weight distribution of polymers poly(dimethyl)siloxane 2.25 kd 37

38 Summary - MALDI Advantages Relatively gentle ionization technique. Very high MW species can be ionized. Molecule need not be volatile. Very easy to get femtomole sensitivity. Usually 1-3 charge states, even for very high MW species. Positive or negative ions from same spot. Disadvantages MALDI matrix cluster ions obscure low m/z (<600) range. Analyte must have very low vapor pressure. Pulsed nature of source limits compatibility with many mass analyzers. Coupling MALDI with chromatography is very difficult. Analytes that absorb laser light can be problematic. 38