Mass Spectrometry and Proteomics - Lecture 1 - Matthias Trost Newcastle University matthias.trost@ncl.ac.uk
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
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
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
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
History of mass spectrometry 1886 E. Goldstein discovers anode rays in a gas discharge tube. 1897 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. 1918 A.J. Demster develops the electron impact ion source and constructs the first mass spectrometer that allows focusing of ions in direction. 1919 F.W. Aston constructs the first mass spectrometer that allows focusing of ions by velocity (Nobel Prize 1922). 1931 E. O. Lawrence invents the cyclotron (Nobel Prize in 1939). 1934 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. 1934 W.R. Smythe, L.H. Rumbaug and S.S. West perform the first preparative separation of isotopes. 1940 A.O. Nier et al isolate the isotope 235 U. 6
History of mass spectrometry 1942 First commercial sector mass spectrometer by CEC. 1948 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 1946. 1952 Quasi-equilibrium theorie (QET) and Rice Ramsperger Kassel Marcus (RRKM) theory explain the molecular fragmentation of ions. Marcus receives Nobel Prize in 1992 1952 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 1989. 1956 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. 1966 M.S.B. Munson et F.H. Field introduce the chemical ionisation. K. Biemann et al determine first peptide sequence by mass spectrometry. 1967 F.W. McLafferty and K.R. Jennings introduce collision-induced dissociation (CID). 1968 Finnigan commercialises the first quadrupol mass spectrometer. 7
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. 1974 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. 1978 R.A. Yost and C.G. Enke construct the first triple quadrupol mass spectrometer. 1981 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 1982. 1982 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 2002. 1988 J. Fenn develops electrospray ionisation after a concept proposed by M.Dole in 1968. Fenn receives Nobel Prize in 2002. 1999 A.A. Makarov presents a new type of mass analyser the Orbitrap. 2004 D.F. Hunt lab develops electron-transfer dissociation (ETD) mass spectrometry. 8
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
The basics of mass measurements: vacuum Vacuum technology Pressure (mbar) Pressure (mtorr) Vacuum 1000-1 750 torr-750 mtorr Primary Vacuum 10 0-10 -3 750-0.75 Intermediate Vacuum 10-3 -10-7 0.75-7.5 * 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 (200-500 L/sec) (20-90,000 rpm!, up to several thousand km/h!). Less used are diffusion pumps (600-2000 L/sec) and cryo-pumps. 10
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
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: 219.67 Da Nominal mass: 219 u Monoisotopic mass: 219.0563 u m/z: 220.06 Th 12
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: 12.0000u <-> 13 C: 13.0034u 35 Cl: 34.9689u <-> 37 Cl: 36.9659u 1u=1 Da=1/12 of 12 C ~ mass of the H-atom (1.00794u) Each isotope has a natural abundance: E.g. 12 C: 100% <-> 13 C: 1.08% 13
The basics of mass measurements: isotopes Symbol #atomic Nominal mass Isotopic Composition Isotopic mass Average Mass H 1 1 100 1.007825 1.00795 2 0.0115 2.014101 Na 11 23 100 22.989769 22.989769 P 15 31 100 30.973762 30.973762 C 6 12 100 12.000000 12.0108 13 1.08 13.003355 N 7 14 100 14.003070 14.00675 15 0.369 15.000109 O 8 16 100 15.994915 15.9994 17 0.038 16.999132 18 0.205 17.999116 S 16 32 100 31.972071 32.067 33 0.8 32.971459 34 4.52 33.967867 36 0.02 35.967081 Cl 17 35 100 34.968853 35.4528 37 31.96 36.965903 Br 35 79 100 78.918338 79.904 81 97.28 80.916291 14
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 2. http://pprco.tripod.com/sims/theory.htm http://nsb.wikidot.com/pl-9-8-3-9 15
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
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) 505.3506 505.3506 100 +1 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) 915.4818 915.7363 915.7363 00 915.4818 protein_modeling TOF MS ES+ 1.86e3 506.3584 506.3584 915.9765 915.9765 +4 507.3566 507.3566 0 500 501 502 503 504 505 506 507 508 509 510 511 512 m/z % 915.2247 915.2274 916.2311 916.2311 0.25 916.4857 916.4857 mix of 6 proteins prot_mix_0724a 655 (10.923) Sm (SG, 2x6.00); Cm (645:675) 100 1086.0433 1086.0433 LCT 1086.5515 1086.5515 protein_modeling TOF MS ES+ 454 m/z 915 916 917 918 0.5 1086.0444 1087.0444 +2 % 916.7402 0 1087.5529 1087.5529 1088.0460 1088.0460 0 m/z 1084 1085 1086 1087 1088 1089 1090 17
Resolution The basics of mass measurements: resolution 18
The basics of mass measurements: resolution Resolution How does the isotopic pattern vary with resolution for a peptide of 2000 Da? 19
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 + 2 249 249.0580 249.0700 249.1479 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
The basics of mass measurements: Accuracy and Precision 21
Ionisation techniques Electrospray ionisation (ESI) Matrix-assisted laser desorption/ionisation (MALDI) Not covered: Electron Ionisation (EI), Chemical Ionisation (CI), Fast-Atom Bombardment (FAB) 22
Electrospray 23
Electrospray 24
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
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
How to determine the molecular mass of a protein from an ESI-MS spectrum 808.3 848.7 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 1 +1 771.6 893.3 738.1 942.8 707.4 998.1 1060.5 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 = 998.1 and m/z 1 = 1060.5 n 1 = 17, and M = 18011 Da 27
Deconvolution of ESI mass spectra Charge states Deconvoluted spectrum M = n(m n H) 3000 30000 Mass (Da) 28
Nobel Prize in Chemistry 2002 2010) For the development of Electrospray Ionisation For the development of Desorption Ionisation Michael Karas Franz Hillenkamp 29
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
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
Matrix-assisted Laser Desorption/Ionisation (MALDI) 1: peptides, 2: proteins, 3: oligosaccherides, 4: Nucleic acids, 5: polymers 32
Matrix-assisted Laser Desorption/Ionisation (MALDI) 33
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
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
Applications: Mass determination of intact proteins Intens. [a.u.] x104 [M+2H] 2+ 8798.5 DHAP_forhighermasses_plusMax 0:G22 MS Raw 5 8798.50 4 3 [M+3H] 3+ 2932.83 2 1 5865.7 [M+H] + 17597.0 0 4000 6000 8000 10000 12000 14000 16000 18000 m/z MALDI-TOF spectrum of a single protein 36
Applications: Molecular weight distribution of polymers poly(dimethyl)siloxane 2.25 kd http://www.arkat-usa.org/?view=manuscript&msid=869 37
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