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Home , Penning trap, Sector mass spectrometer

Mass Spectrometry and Proteomics - Lecture 1 -

Matthias Trost Newcastle University [email protected] Content Lectures 1-3

• The basics of measurement Lecture 1 • Ionisation techniques • Mass analysers • Detectors Lecture 2 • Tandem • Fragmentation techniques Lecture 3 • Peptide fragmentation • Hybrid instruments

2 Content Lectures 4-6

• What is proteomics?

• Sample Preparation Lecture 4 • Experimental Design • Quantification techniques • Search engines, Databases, FDR Lecture 5 • Data analysis & Data inspection • Fractionation techniques Lecture 6 • Phosphoproteomics and other PTMs • Proteomics experiments

3 Lecture 1

• Basics – Components of a mass – 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 “”.

5 History of mass spectrometry

1886 E. Goldstein discovers anode rays in a gas discharge tube. 1897 J.J. Thomson discovers the and determines its m/z (Nobel Prize in 1906)

1912 J.J. Thomson constructs the first mass spectrometer and sees spectra of O2, N2, CO, CO2 and COCl2. He observes negative , multiply charged ions and identifies isotopes of 20Ne and 22Ne. 1918 A.J. Demster develops the electron impact 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 (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 235U.

6 History of mass spectrometry

1942 First commercial 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 mass spectrometer. W. Paul, H.S Reinhard and U. von Zahn publish the first quadrupol mass spectrometer. Paul and Dehmelt (“”) 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 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 . 2004 D.F. Hunt lab develops electron-transfer dissociation (ETD) mass spectrometry.

8 The basics of mass measurements

Sample IonisationMass analyser Detector Data introduction Acquisition and Analysis

Electron ESI Quadrupol multiplier Computer MALDI Ion trap Microchannel Chromatography plate (GC, HPLC) FAB TOF Ion-to-photon Direct injection CI FT-ICR detectors Capillary FD/FI Sectors (B, E) FT-ICR electrophoresis II Orbitrap Orbitrap

Vacuum

9 The basics of mass measurements: vacuum

Vacuum technology Pressure (mbar) Pressure (mtorr) Vacuum 1000 - 1 750 torr-750 mtorr Primary Vacuum 100-10-3 750-0.75 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 m3/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 Å2 for a small peptide ion). Using a collision cross section of 50 Å2, the following table may be constructed:

11 The basics of mass measurements

Positive-ion mode: the molecule with an additional Negative-ion mode: the molecule with a proton less. The calculated mass can be obtained from the empirical

formula: C11H10N3Cl In the literature, the molecular mass can be shown as: Average mass: 219.67 Da Normalised on the most abundant peak Nominal mass: 219 u : 219.0563 u m/z: 220.06 Th 12 The basics of mass measurements: isotopes 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: 12C: 12.0000u <-> 13C: 13.0034u 35Cl: 34.9689u <-> 37Cl: 36.9659u 1u=1 Da=1/12 of 12C ~ mass of the H-atom (1.00794u) • Each isotope has a natural abundance: E.g. 12C: 100% <-> 13C: 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 (, neutrons and ). 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=mc2.

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 13C-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 protein_modeling prot_mix_0724a 651 (10.856) Sm (SG, 2x6.00); Cm (648:651) 505.3506 TOF MS ES+ 505.3506 783 100 +1

% 1.00 mix of 6 proteins LCT protein_modeling rot_mix_0724a 350 (5.837) Sm (SG, 2x6.00); Cm (343:374) 915.7363 TOF MS ES+ 506.3584 915.4818 915.7363 1.86e3 506.3584 00 915.4818 507.3566 915.9765 507.3566 915.9765 +4 0 m/z 500 501 502 503 504 505 506 507 508 509 510 511 512 915.2247 % 915.2274 916.2311 916.2311

mix of 6 proteins LCT protein_modeling prot_mix_0724a 655 (10.923) Sm (SG, 2x6.00); Cm (645:675) 1086.5515 TOF MS ES+ 916.4857 1086.5515 454 0.25 916.4857 100 1086.0433 916.7402 1086.0433

0 m/z 915 916 917 918 0.5 1086.0444 1087.0444 +2 %

1087.5529 1087.5529 1088.0460 1088.0460

0 m/z 1084 1085 1086 1087 1088 1089 1090 17 The basics of mass measurements: resolution 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:

+ C20H9 + R>10000 R=1000 C19H7N

C H N O + + + + 13 19 3 2 C19H7N C20H9 C13H19N3O2

249 249.0580 249.0700 249.1479

Typical resolution of mass : • 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

n+ 848.7 • Observed ions have composition [M+nH] • For the charge states of m/z1 (higher value) 808.3 and m/z2 (lower value) we have n2=n1+1

771.6 893.3 •m= m/z = (H=mass of proton) n 1 738.1 942.8 • Its neighbouring peak to the left: 707.4 •mn+1=m/z2 = = 998.1 (with M being the mass of the protein) • 1060.5 Solving both equations for n and M:

/ •n1 = // •M = n1(m/z1 –H) m/z • e.g. m/z2 = 998.1 and m/z1 = 1060.5 n must be an integer •n1 = 17, and M = 18011 Da 27 Deconvolution of ESI mass spectra

Deconvoluted Charge states spectrum

M = n(mn –H)

3000 30000 Mass (Da)

28 Nobel Prize in Chemistry 2002

2010) Michael Karas Franz Hillenkamp For the development of For the development of Electrospray Ionisation Desorption Ionisation

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

80x magnification of dried Sample target 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

200Wavelength (nm) 500

Common lasers; N2 (337 nm), ArF excimer (193), Nd-YAG frequency tripled (355 nm) and quadrupled (266 nm) 35 Applications: Mass determination of intact proteins

x104 DHAP_forhighermasses_plusMax 0:G22 MS Raw [M+2H]2+

Intens. [a.u.] 8798.5 5

8798.50

4

3 [M+3H]3+

2932.83

2 [M+H]+ 17597.0 1 5865.7

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

38