Ion Sources and Mass Analyzers in Protein Characterization

Principles of MS and MS/MS

Matrix Assisted Laser Desorption Ionization (MALDI)Electrospray Ionization (ESI), Nano-ESI

Time of FlightQuadrupole Mass Filter

Quadrupole Ion TrapFourier Transform Ion Cyclotron Resonance

Mass Spectrometry

Lenses and prisms focus and refract light.

Analogous systems can focus and deflect ions in a vacuum.

1. Get molecules into the gas phase & ionize them.2. Give the ions a defined energy or velocity.3. Separate or sort the ions on the basis of that defined property.4. Detect the ions & assign their masses.

A very simple mass spectrum of Carbon Dioxide

Copyright ASMS: http://www.asms.org/whatisms/p5.html

Online Separations with MS Detection

Sensitivity, Specificity, Transparency of DataDifferentiation of Co-eluting analytes

Looking at MS Data: LC/MS Data is Three Dimensional

Mass spec data systems generate “total ion chromatograms” by integrating spectra and plotting intensity versus time. It is analogous to that generated using a diode array UV-detector on an HPLC system. The data is fundamentally 3-dimensional.

A “selected ion chromatogram” is the same graph of intensity over time for a defined m/z. It is analogous to a UV chromatogram for a single wavelength.

Looking at MS Data: Mass spectra show m/z, not mass

Mass spectrometers separate molecules on the basis of their mass to charge ratio, not their mass. That means the x-axis is not necessarily reflective of M.

Mass spectra are normalized to the abundance (intensity) of the highest peak in a given spectrum. The y-axis is always scaled from 0-100. Absolute intensity is also often shown in the corner of the spectrum as an arbitrary number unique to each data system.

Mass Resolution

dM

FWHM 25% valley

M1 M2 Resolution is often defined as M/dM.

“Unit resolution” means that two adjacent peaks are resolved from one another.

In low resolution, dM may be 1 mass unit.

In high resolution, dM may be 0.010 mass unit.

However, the actual resolution depends on how one defines the separation between the peaks (e.g. 50% vs 10% valley).

Larger Peptides = More Complex Isotope Patterns

As ions grow larger, the “12C” peak is not necessarily most abundant.

The mass resolution of analyzers may not always be adequate to distinguish individual peaks. In this case, average masses are used.

It is important to be aware of the capabilities of the mass analyzer one is using.

Average mass:The mass of an ion for a given empirical formula calculated using the relative average atomic mass of each element,e.g. C = 12.01115, H = 1.00797, O = 15.9994.

Monoisotopic mass:The mass of an ion for a given empirical formula calculated using the exact mass of the most abundant isotope of each element,e.g., C = 12.000000, H = 1.007825,O = 15.994915.

Analyzer Resolution: Average vs. Monoisotopic Masses

Mass Spectrometer

Tandem Mass Spectrometer

Tandem MS permits selection and isolation of specific ions for subsequent analysis.

Tandem instruments have multiple mass analyzers.

Tandem Mass Spectrometry (MS/MS)

Magnetic Sector and Double Focusing Instruments

Quadrupole Mass Filters

Quadrupole Ion Traps

Fourier Transform Ion Cyclotron Resonance

Time of Flight

Mass Analyzers

Mass Analyzers: The Quadrupole Mass FilterA potential of ~100-1000 V is applied alternately to the opposing pairs of rods at a frequency of a few MHz. At a specific combination of DC & RF, an m/z has a stable trajectory through the rods, and all other m/z are lost. The mass range is scanned as the voltages are swept from min to max, but at constant DC/RF ratio.

Faster Scanning than sector instruments (but not as fast as ion traps or TOF).Mass Range generally m/z 0-2000 or 0-4000.Facile MS/MS using Triple Quadrupole (Q-q-Q) analyzer.Exquisitely sensitive in selected ion monitoring (both analyzers parked at one m/z).Largely replaced by the ion trap and hybrid Q-q-TOF for biopolymer analysis.

MS/MS in a Triple Quadrupole (Q-q-Q) Mass Spectrometer

Facile MSn

High resolution over narrow rangesExtremely SensitiveFast ScanningSmallInexpensive

Mass Analyzers: The Quadrupole Ion Trap

Mass Analyzers:Fourier Transform Ion Cyclotron Resonance

Ions in a magnetic field move in circular orbits characteristic of their m/z values. If energy is provided at a frequency equal to their precession frequency, and in a direction perpendicular to their plane of precession, the ions will absorb the energy, enabling them to be detected.

Extremely High Resolution

MSn capability

Must Operate at very good vacuum

Superconducting Magnet

Difficult to operate

Becoming increasingly reliable

Mass Analyzers:Fourier Transform Ion Cyclotron Resonance

Linear TOF

Reflectron TOF

Constant Kinetic EnergyzeV = ½ mv2 v = (2zeV/m)½

Mass Analyzers: Time of Flight (TOF)

Ion Sources

Gas Phase Ionization:Electron Impact (EI)Chemical Ionization (CI)

Desorption Ionization:252Cf Plasma Desorption (PDMS)Fast Atom Bombardment (FAB) / Secondary Ion MS (SIMS)Laser Desorption (LDMS)Matrix Assisted Laser Desorption (MALDI)

Spray Ionization:Thermospray (TSP)Atmospheric Pressure Chemical Ionization (APCI)Electrospray (atmospheric pressure ionization) (ESI, API)

http://www.nobel.se/chemistry/laureates/2002/index.htmlJohn B. Fenn – Nobel Lecture"Electrospray Wings for Molecular Elephants" http://www.nobel.se/chemistry/laureates/2002/fenn-lecture.html

John B. Fennelectrospray ionization for MS

Koichi Tanakasoft laser desorption ionization for MS

Kurt Wuthrichsolution NMR for protein structures

The Nobel Prize in Chemistry 2002

MALDI-TOFMS

Analyte:10 – 1000 fmol1 – 500 kDa

MALDI-TOFMS

the three most commonly used matrices

Some Characteristics of MALDI-TOFMS

Ions are easy to generate

Buffers, salts, some detergents easily tolerated

Excellent sensitivity (< 20 fmol for digests)

High resolution at low mass with time lag focusing

Resolution drops off at higher mass (>20 kDa)

Protein or peptide mixtures can show suppression effects

Different matrices yield different results

A MALDI Target with Digest Samples Spotted on Nitrocellulose Films

R. G. Davis, GlaxoSmithKline

MALDI-TOFMS

Constant Kinetic EnergyzeV = ½ mv2 v = (2zeV/m)½

Ion Sources: ElectrosprayVery gentle and efficient way of getting gas phase ions from solutions.

A fine spray of charged droplets is generated in an electric field.

Droplets evaporate - analyte molecules are left carrying charges.

Multiply Charged Ions are the rule.

Concentration dependent – High sensitivity at very low flow rates (<< 1 ul/min).

Electrospray is a concentration-dependent technique.Lower flow rates are favored significantly.

Smith et al, Acc. Chem. Res. 2004

Quasimolecular ions, [M+nH], from myoglobin, Mr= 16,951.5 Da.

Using adjacent pairs of ions, the molecular mass of the myoglobin can be calculated very accurately.

+21+12

m1 = (M+n)/nm2 = (M+n+1)/(n+1)

Electrospray Mass Spectrum of Myoglobin

Tandem Mass Spectrometry (MS/MS) is the Method of Choice for Sequence Analysis of Peptides

SpeedSensitivity

Tolerance for Amino-terminal Blocking GroupsHigh Specificity for Protein Identification

Mass Spectrometer

Tandem Mass Spectrometer

Tandem MS permits selection and isolation of specific ions for subsequent analysis.

Tandem instruments have multiple mass analyzers.

Tandem Mass Spectrometry (MS/MS)

Tandem Mass Spectrometry : Product Ion Scan

Q1 Q2 Q3MASS FILTER RF ONLY MASS FILTER

PRECURSOR IONSELECTION

NEUTRAL GASCOLLISIONS

PRODUCT IONDETECTION

ION SOURCEDETECTOR

1. “Parent” Ions are selected and isolated2. Collision-Induced-Dissociation Results in fragmentation3. “Daughter” Ions are characterized with the second mass analyzer

Tandem Mass Spectrometry: Precursor Ion Scan

Q1 Q2 Q3MASS FILTER RF ONLY MASS FILTER

PRECURSOR IONSELECTION

NEUTRAL GASCOLLISIONS

PRODUCT IONDETECTION

ION SOURCEDETECTOR

1. “Product” Ion is selected and Q3 is parked2. Q1 is scanned normally3. Only precursors which fragment to produce selected product ion are detected.

Tandem Mass Spectrometry: Neutral Loss Scan

Q1 Q2 Q3MASS FILTER RF ONLY MASS FILTER

PRECURSOR IONSELECTION

NEUTRAL GASCOLLISIONS

PRODUCT IONDETECTION

ION SOURCEDETECTOR

1. The mass of a functional group whose loss is to be detected is selected.2. Both Q1 and Q3 are scanned simultaneously, offset by the selected “neutral loss” mass.3. Collision-Induced-Dissociation Results in fragmentation4. Daughter” Ions are detected only when the specified loss occurs in Q2,

indicating the presence of the moiety of interest.

Micromass “Back to Basics” http://www.micromass.co.uk/basics/index.html

MS/MS of Angiotensin III:selection and fragmentation of the (M+H)+ molecular ion at m/z932

Micromass “Back to Basics” http://www.micromass.co.uk/basics/index.html

MS/MS of Angiotensin III:selection and fragmentation of the (M+H)+ molecular ion at m/z932

532 669 784

400

Another way to label an MS/MS spectrum is to draw lines through the structure, with pointers indicating which part of molecule is being detected following fragmentation. These markers may be labeled with masses.

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

0

100

%

Y;136.1

L86.1

y''10 2+

643.4

y''9 2+578.8a1

197.1

558.3

y''7 2+457.8293.1

y''11 2+

724.9

643.9

z5644.4

z7897.5757.9

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700mass0

100

%

y''11

y''10

1285.7

Y136.1

L86.1

y''9

1156.7

a1197.1

y''7

914.6265.1

b2388.2

y''4505.3 643.3 781.4

y''8

1042.7

z101268.69

z111431.8 1672.9a11

1513.81655.8

MaxEnt-3TM

Raw data

1448.8

MaxEnt-3TM for Sequencing

Links to Information on Mass Spectrometry

Information on FTICR at the national high magnetic field labhttp://www.nhmfl.gov/science/cimar/icr/

Introduction to mass spectrometry at SciMedia.comhttp://www.rmsb.u-bordeaux2.fr/rmsb/ms/IntroMS.html

The Thermo Finnigan homepagehttp://www.thermo.com/eThermo/CDA/BU_Home/BU_Homepage/0,12482,113,00.html

The Micromass homepage, Mass Spec Back to Basics coursehttp://www.micromass.co.uk/basics/default.asp

Mass Spec Glossaryhttp://www.genomicglossaries.com/content/mass_spectrometry.asp

The I-mass homepagehttp://www.i-mass.com/

I-mass tutorialshttp://www.i-mass.com/guide/tutorial.html

American Society for Mass Spectrometry: What is Mass Spectrometryhttp://www.asms.org/whatisms/