What Is a Mass Spectrum? 1265.6038 100

Outline EMBnet course 28.1.-1.2.2008 Day 1 Mass spectrometry in proteomics Mass spectrometry, general introduction

What are the constituents of a mass spectrometer

How the instruments look like

Pierre-Alain Binz

Swiss Institute of Bioinformatics, proteome informatics group Geneva Bioinformatics SA (GeneBio) EMBnet course Basle, 28 Jan, 2008

What is a mass spectrum? 1265.6038 100

MALDI-DE-RE-TOF MS 80 1394.7169 tryptic digest of BSA

1252.6472 1757.8374

870.4042 1299.6103 * % Intensity 40 * 1930.0053 1742.8780 * 1410.7018 *

1787.7116 2062.0077 950.4584 2523.2021 20 * 1083.5082 1778.0565 2848.3 2285.1 2467.1695 848.2 1099.5 1859.9261 2065.0 2266.1 1364.7 1555.7 2501.3228 2016.0 2222.2043 2734.2 0 828.0 1263.2 1698.4 2133.6 2568.8 3004.0 Mass (m/z)

Protein Identification using Mass Spectrometry Outline

protein from gel/ tryptic digestion & Mass spectrometry, general introduction 1-DE, 2-DE, PVDF/LC fraction peptide extraction LC TYGGAAR EHICLLGK GANK PSTTGVEMFR unmodified and What is a mass spectrum Mass spectrometry, modified peptides peptide mass fingerprints What are the constituents of a mass spectrometer

PMF identification How the instruments look like MS Fragmentation

Mass spectrometry, MS/MS identification peptide MS fragments

1 What is a mass spectrum? How does a peptide signal looks like? 1265.6038 100

MALDI-DE-RE-TOF MS 80 1394.7169 tryptic digest of BSA Low resolution

1252.6472 1757.8374

870.4042 1299.6103 * % Intensity 40 * 1930.0053 1742.8780 * 1410.7018 *

1787.7116 2062.0077 High resolution 950.4584 2523.2021 20 * 1083.5082 1778.0565 2848.3 2285.1 2467.1695 848.2 1099.5 1859.9261 2065.0 2266.1 1364.7 1555.7 2501.3228 2016.0 2222.2043 2734.2 0 828.0 1263.2 1698.4 2133.6 2568.8 3004.0 Mass (m/z)

Isotopic distribution Isotopic distribution

Mass resolution 0.1% vs. 1 ppm

Symbol Mass Abund. Symbol Mass Abund ------

C(12) 12.000000 98.90 C(13) 13.003355 1.10 N(14) 14.003074 99.63 N(15) 15.000109 0.37 O(16) 15.994915 99.76 O(17) 16.999131 0.038 H(1) 1.007825 99.99 H(2) 2.014102 0.015 S(32) 31.972072 95.02 S(33) 32.971459 0.75

Mass resolution Mass resolution

1.0 FWHM 0.7 FWHM 0.5 FWHM 2000

Full width

1000 0.3 FWHM 0.2 FWHM 0.1 FWHM

Half mass

2 524.3 262.6 100 100

95 95 90 90 85 85 80 80 75 Singly charged Ion: 75 Doubly charged Ion: 70 70 65 Distance between Peak 65 Distance between Peak and 60 and Isotop 1 amu 60 Isotop 0.5 amu 55 55 50 50 45 45 ∆ = 1.0 amu ∆ = 0.5 amu Relative Abundance Relative Abundance 40 40

35 35 30 30 525.3 25 25 ∆ = 1.0 amu 263.1 20 20 ∆ 15 15 = 0.5 amu 10 10 526.2 263.6 5 5 0 0 520 521 522 523 524 525 526 527 528 529 258 259 260 261 262 263 264 265 266 267 m/z

m/z

Resolution: Example Peptide Mw 2129.64, Ion 4+ M ultiply charged myoglobin ions from ES I

Intens. (M2-1.008) /M1-M2 = Z1 x105 1060.5 533.46 100 M1 (Z1 * M1)-(Z*1.008) = Mwt 90 1131.1 Resolution 998.2 1211.9 4 M2 942.9 0.6 m/z 80 1305.0 2 70 893.3 60 848.6 1413.5 0 531 532 533 534 m/z 50

40 808.2 Intens. 1541.9 x105 532.62 532.85 30 771.5 20 1696.0 533.09 Resolution 616.2 738.1 1.0 10 707.3 1310.9 1884.2 1428.7 1563.0 1820.8 1888.9 533.33 0.2 m/z 0 0.5 600 800 1000 1200 1400 1600 1800 2000 533.61 m/z 0.0 531 532 533 534 m/z

9.9E+3 MALDI-DE-RE-TOF MS 100 90 tryptic digest of BSA 80 1265.6038 100 Deconvoluted myoglobin spectrum 70

50 16951.0 100 80 1394.7169 Intensity % 40 90

30 80

20 70 60

60 1252.6472 10 1757.8374

50 0 0 870.4042 1299.6103 1910.0* 1918.8 1927.6 1936.4 1945.2 1954.0

40 % Intensity 40 * Mass (m/z) 30 1930.0053 1742.8780 * 1410.7018 * 20 1787.7116 2062.0077 10 950.4584 2523.2021 15931.0 16104.0 16392.0 16582.0 16784.0 17088.0 17280.0 17562.0 17830.0 17995.0 20 * 1083.5082 2848.3 0 1778.0565 2285.1 16000 16200 16400 16600 16800 17000 17200 17400 17600 17800 18000 2467.1695 848.2 1099.5 1859.9261 2065.0 2266.1 mass 1364.7 1555.7 2501.3228 2016.0 2222.2043 2734.2 0 828.0 1263.2 1698.4 2133.6 2568.8 3004.0 Mass (m/z)

3 HPLC-ESI-autoMS/MS Ion fragmentation with Mass Spectrometry Int. H I 7 x10 TIC O O 4

H 2 I N O Tandem MS or MS/MS 0 4.0 5.0 Time [min] HO Ab. MS, Time=4.420min 100 634 m/z 634

One set of ions (one m/z value) is selected from a mixture of ions; 50 MS/MS These ions are fragmented; the fragments are measured. 545 0 100 200 300 400 500 600 m/z H I O Ab. MS/MS(634), Time=4.458min 545 OH 100 373

I 50 249 376 563 O HO 0 m/z 563 100 200 300 400 500 600 m/z

Peptide fragmentation with MS/MS Outline

Mass spectrometry, general introduction

What is a mass spectrum

What are the constituents of a mass spectrometer K C S C N P D M MAPNCSCK 2+ y3 [M+2H] MAPNCSC K y1 How the instruments look like MAPNCS CK y7 y4 y5 MAPNC SCK y2 y6 MAPN CSCK y8 ...

How are mass spectra produced ? Generic description of a mass spectrometer • Ions are produced in the source and are transferred into the mass analyser Atmosphere Vacuum System

Sample Ionisation Mass Data • They are separated according to their mass/charge Detector Inlet Method Analyser System ratio in the mass analyser (e.g. Quadrupole, Ion Trap, Time of Flight)

• Ions of the various m/z values exit the analyser and are ‘counted’ by the detector

4 EI electron impact ionisation: beam of electrons through the gas-phase sample. Produces Ionization methods molecular ions or fragment ions. Typically 70eV. Sample heated. Ionization methods + Reproducible, structural information Analytes are ionized to be driven in the mass analyzer - sample must be volatile and stable, molecular ion often abscent mass range: < 1000Da

Electron impact (EI) CI: chemical ionisation: reagent gaz (methane, isobutane, or ammonia) ionized with electrons. High gaz pressure: (R = reagent, S = sample, e = electron, . = radical electron , H = hydrogen) Chemical Ionisation (CI) R + e ---> R+. + 2e Fast atom bombardment (FAB) R+. + RH ---> RH+ + R. RH+ + S ---> SH+ + R Field desorption (FD) Heated sample. Atmospheric Pressure Chemical Ionisation (APCI) + [M+H]+ often visible, less fragmentation than EI - sample must be volatile and stable, less structural info than EI mass range: < 1000Da ESI Electro-Spray Ionization DCI: Desorption CI : CI on a heated filament MALDI Matrix Assisted Laser Desorption Ionization + rapid, simple - reproducibility mass range <1500Da

NCI: negative-ion CI: electron capture; use of Methane to slow down electrons + efficient, sensitive; less fragmentation that EI, CI - not all molecule compatible, reproducibility mass range <1000Da

FD: Field Desorption: sample deposited on filament gradually heated by electric field. ESI: electrospray ionisation: The sample solution is sprayed across a high potential Sample ionise by electron tunneling. Ions are M+ and [M+Na]+ difference (a few kilovolts) from a needle into an orifice in the interface. + simple spectra, almost no background Heat and gas flows are used to desolvate the ions existing in the sample solution. - sensitive to alkali, slow, volatile to desorb ESI often produces multiply charged ions with the number of charges tending to increase mass range <2000-3000Da as the molecular weight increases. High to low flow rates 1 ml/min to nl/min. FI: Field ionisation: sample introduced in gas phase (heaten or not), ionised by electron + good for charged, polar or basic compounds, m/z ok for most MS, best for multiply tunneling near the emitter. charged ions, low background, controlled fragmentation, MS/MS compatible + simple spectra, almost no background - complementary to APCI: not good for uncharged, non-basic, low-polarity compounds, - sample must be volatile low ion currents mass range <1000Da mass range <200’000Da

FAB: fast atom bombardment: analyte in a liquid matrix (glycerol, etc.). Bombardment with APCI: atmospheric pressure CI: as in ESI, sample introduced in a high potential fast atom beam (xenon at 6keV). Desorbtion of molecular ions, fragments and matrix difference field. Uses a corona discharge for better ionisation of less polar molecules than clusters in ESI. APCI and ESI are complementary sample introduced liquid, or LC/MS + rapid, simple, good for variety of compounds, strong currents, high resolution MALDI: Matrix-Assisted Laser Desorption Ionization: analyte co-crystallised in matrix. - background, sample must be soluble in matrix The matrix chromophore absorbs and distribute the energy of a laser, produced a plasma, mass range ~300-6000Da vaporates and ionize the sample. + rapid, convenient for molecular weight (singly charged ions mostly) SIMS: soft ionisation: similar to FAB but with ion beam as gas (Ce+), allowing higher - MS/MS difficult, almost not compatible with LC coupling acceleration (energy) <500’000Da + idem FAB - idem FAB, target can get hotter, more maintenance mass range 300-13000Da

Matrix Assisted Laser Desorption/Ionization Electrospray Ionization (ESI) MALDI

UV or IR laser

pump sample S S + + S S SH + + + + + + MH + S + + + + S MH SH MH target +++ + 2+ Membrane, gel or metal + + S + + 2+ S + S SnH MH2 MH2 S + Matrix Smaller Coulomb explosion: Ions grid droplet droplet Clusters and Analytes ionic species Modif. From Alex Scherl

5 Matrix Assisted Laser Desorption/Ionization MALDI Mass Analyzers

Mass Spectrometers separate ions according to their mass-to- charge (m/z) ratios

– Magnetic Sector – Quadrupole – Ion Trap – Time-of-flight – Hybrid- Sector/trap, Quad/TOF, etc.

Time of Flight (TOF) mass analyzer Ion source High vacuum High vacuum Ion source flight tube flight tube

Detector Detector

time 1 Small ions are faster than Reflectron heavy, and reach detector time 2 first

time 3

Quadrupole mass analyzer Ion Trap mass analyzer

• Consists of ring electrode and two + + end caps + • Principle very similar to quadrupole • Ions stored by RF & DC fields + • Scanning field can eject ions of RF + DC specific m/z The quadrupole consists of • The ion is transmitted along the •Advantages two pairs of parallel rods with quadrupole in a stable trajectory Rf field. • - MS/MS/MS….. applied DC and RF voltages. The ion does not have a stable • - High sensitivity full scan Ions are scanned by varying trajectory and is ejected from the MS/MS the DC/Rf quadrupole quadrupole. voltages.

6 Linear Trap Hybrid Mass Spectrometers

3D Trap Full Scan Sensitivity Linear Trap 3 MS (or greater) Tandem in Tandem in SPACE & TIME TIME

QqQ Full Scan Sensitivity 3 MRM Sensitivity MS Neutral Loss MRM Sensitivity Precursor Scan Neutral Loss Tandem in Precursor Scan SPACE + Novel Scan Types

From K Rose

FTMS

Ions moving at their cyclotron frequency can absorb RF energy at this same frequency. A pulse of RF excites the ions in the magnetic field. The ions re-emit the radiation, which is picked up by the reciever plates. The decay produces a free-induction decay signal that can be Fourier transformed to produce the emitted frequencies, and therefore the masses of the ions present.

FTMS Ion Motion in Orbitrap • Only an axial frequency does not depend on initial energy, angle, and position of ions, so it can be used for mass analysis • The axial oscillation frequency follows the formula

k w = m / z

w = oscillation frequency k = instrumental const. m/z = …. what we want!

A.A. Makarov, Anal. Chem. 2000, 72: 1156-1162. A.A. Makarov et al., Anal. Chem. 2006, 78: 2113-2120.

7 Ions of Different m/z in Orbitrap How Big Is Orbitrap? • Large ion capacity - stacking the rings • Fourier transform needed to obtain individual frequencies of ions of different m/z

Outline MS instruments used in Proteomics Mass spectrometry, general introduction ESI-Triple quadrupole MS ESI-Q-TOF MS What is a mass spectrum ESI-Ion-trap MS ESI-Q-trap MS ESI-FTICR MS What are the constituents of a mass spectrometer ESI-LTQ-Orbitrap

SELDI MS How the instruments look like MALDI-TOF MS MALDI-TOF-TOF MS MALDI-Q-TOF MS MALDI-Ion-trap MS MALDI-FTICR MS…

MALDI-TOF MS: illustrated examples MALDI sample plates MALDI-TOF-MS

Voyager STR LASER Voyager DE-PRO Applied Biosystems Applied Biosystems

m/z Autoflex Reflex III Bruker Bruker

Micromass

8 (ESI) - Triple quadrupole MS Q1 MassProduct selection Ion Q2 collisionScan cell (3Q)Q3 Full Scan A Q2 is Non-Linear Collision Cell C B Ion C C+gas Products Products

D Q0 Q1 Q2 Q3

ESI Probe ions in Q1 only transmits ion CFragment the Ion C Q3 Scans for products source

• #1 scan mode used in proteomics as it generates primary structure (sequence) information • The observed signal is a result of the mass-analyzed product ions derived from a mass-selected precursor ion: low energy collisions Square Rod Ion Transmission Hyperbolic, high Electron to Analytical Quads precision Multiplier, Detection Typical product ion quadrupoles spectrum of a peptide System fragmented under low energy conditions

400 800 1200 m/z

Q1 Mass selection Q2 Q3 Q1 Pass A-H O Q2 collision cell Q3 Pass A Selected Ion Monitoring (3Q) Neutral Loss2 Scan Mode (3Q) A Ions C Ion A-H O A-F 2 B Ion C Ion c Ion B Ion C + A-H2O A A D Ion D +H2O Ion E Ion F ions in Q1 only transmits ion CQ2 only transmits ion C Q3 only transmits ion C source Scans across mass range Fragment ions one at a timeScans across mass • High sensitivity, due to short mass scanning range (can switch) (NB A-bond-HOH, not range at 18 amu lower • We know what we are looking for (ion C and standard) minus) than Q1 (linked scan) • For complex samples (plasma) it is common to have multiple peaks

• The setup of a TSQ can easily be understood: • Q1 and Q2 are scanned with an offset of the neutral loss n to be detected. Thus, Q1 passes m/z(M), M fragments in Q2 by loss of n and Q2 passes m/z(M)-n. One important analytical application of CNL scans is their use in SRM (selected reaction monitoring)

Single/Multiple Reaction Q1 Mass selection Q2 collision cell Q3 pass only 79 Q1 Mass selection Q2 collision cell Precursor Ion Scan Mode (3Q) Monitoring (3Q) Q3 Mass Selection Ions Ion A-PO A-F 4 A SRM/MRM Ion B Ion C C PO3- 79 B Ion C C+gas C1+C2+C3 Ion C2 Ion D Ion E D Ion F

Scans across mass rangeFragment ions one at a time Transmits only 79 Ions in Q1 only transmits ion CFragment Ion C Q3 only transmits ion Source C2 In the precursor ion scan, the instrument looks for a predefined m/z product ion and associates it back to the precursor ion it originated from MRM/SRM is performed by specifying the parent mass(es) of the compound for MS/MS • Example in a negative ion mode: fragmentation and then specifically monitoring for (a single) fragment ion(s) The MS can transmits m/z of -79 (a negatively charged phosphate ion), and identify which peptide ion lost the phosphate ion, thereby identifying it as a phosphopeptide • MRM/SRM can generate fragment ions that can be measured and quantified from very complicated mixtures (e.g. plasma) The mass spectrum reveals all precursor ions that fragment to yield a common product • SRM typically contain a single peak: ideal for sensitive and specific quantitation ion

9 ESI-Q-TOF MS, MS mode ESI-Q-q-TOF, MS/MS mode Q q TOF Q q TOF ESI ESI

I Ion 2 Fragment 2 ¯ Ion 1 I Ion 3 Fragment 1 ¯ Fragment 3

m/z m/z

Mod. From Alex Scherl Mod. From Alex Scherl

Esquire-LC Ion Optics Q-TOF MS

HPLC inlet Skimmers Octopole End Caps

Capillary

Q Star XL Hybrid BioTOF-q qTOF-Ultima Applied Biosystems Bruker Micromass

+ + + + + + + + + + + + + + Ion trap MS

Ion Trap Nebulizer Lenses LCQ Deca XP Esquire 3000 Ring Electrode Finnigan Bruker

nanoLC-ESI-Q-TOF Principe of LC-MS/MS

Q-Tof time 27.4 min : peak at m/z = 957.6 m/z = 957.6 QIIEEDAALVEIGPR Column C18 75 mm Q96DH1 HPLC Autosampler/Injector

10 4700 TOF/TOF from Applied Biosystems MALDI TOF-TOF: Sample Plate MS/MS Mode Reflector Laser Detector Reflector

CID Cell intensity TIS Mass (m/z)

TOF 1 TOF 2 collision Source 2 source cell Source 1 V V 1 2

TOF 1 TOF 2

Timed ion selector operation Bruker UltraFlex TOF-TOF

TIS Deceleration from ion 0+ source

m m m mmm m m123 mmm12 3 mm1m2 3 m1m2m3 1 2 3 1 1 2m2 2 m2 m2 m2 m2 m2 m2 m2 mm31 m3 mm13 mm13 0- TOF 1 mm31

m3 m1 to collision cell Æ

5 V TTL Pulse 0 V

+950 V Switch down Switch up time time calculated TIS 0 V calculated by by low mass Single Gate high mass gate

gate geometry -950 V geometry

Timed ion selector operation

TIS from ion 0+ source

TOF 0- CID TOF 2 LIFT

Switch down Switch up time time calculated calculated by by low mass Few ns high mass gate gate geometry geometry TIS CID Cell V1 V2

11 MALDI TOF-TOF MS nanoLC-MALDI-TOF-TOF AB 4700 Proteomics Analyzer with Auto-loader Spotting robot

Column C18 75 mm

MALDI plate HPLC Autosampler/Injector

TOF-TOF from Bruker: the Ultraflex

Off-line MALDI MS (MS/MS)

ElectroSpray MALDI EI/CI Switchable CF-FAB, CF-SIMS GC Interface LC Interface Pulsed valve for MS/MS IRMPD Bruker APEXIII

Operating mass range (APEX 70e) of 18 - FTMS can provide very high resolution, 106, which its main 66000 Daltons advantage compared to other mass spectrometers. Mass accuracy <1ppm in MS and MS/MS mode

Q Trap (Quadrupole – linear trap) linear-TRAP MS The Q-trap MS

Q-trap LTQ-XL Applied Biosystems Thermo Fisher Scientific and MDS Sciex

12 LTQ Orbitrap Operation Principle

1. Ions are stored in the Linear Trap 2. …. are axially ejected LTQ-Orbitrap 3. …. and trapped in the C-trap 4. …. they are squeezed into a small cloud and injected into the Orbitrap 5. …. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation

The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier

Ions of only one mass generate a sine wave signal

From Thermo

Additional info on MS

http://www.spectroscopynow.com/ http://www.ionsource.com/ http://www.asms.org/whatisms/index.html