Fundamentals of Mass Spec

8/6/2019 Fundamentals of Mass Spec

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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 Trap

Fourier 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.





Online Separations with MS Detection

Sensitivity, Specificity, Transparency of Data

Differentiation 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 alsooften shown in the corner of the spectrum as an arbitrary number unique to each

data system.



Mass Resolution

dM

FWHM 25% valley

M1 M2Resolution is oftendefined 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 notnecessarily 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 formulacalculated 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 specificions 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


Time of Flight

Mass Analyzers



Mass Analyzers: The Quadrupole Mass Filter

A 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. Themass 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 ranges

Extremely Sensitive

Fast ScanningSmall

Inexpensive

Mass Analyzers: The Quadrupole Ion Trap



Mass Analyzers:


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:






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. Fenn

electrospray ionization for MS

Koichi Tanaka

soft laser desorption ionization for MS

Kurt Wuthrich

solution 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: Electrospray

Very 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)/n

m2 = (M+n+1)/(n+1)

Electrospray Mass Spectrum of Myoglobin





Mass Spectrometer

Tandem Mass Spectrometer

Tandem MS permits

selection and

isolation of specificions 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 isolated

2. Collision-Induced-Dissociation Results in fragmentation3. “Daughter” Ions are characterized with the second mass analyzer



T d M S t t N t l L S



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.



MS/MS f A i i III



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 tolabel an MS/MS

spectrum is to

draw lines

through the

structure, withpointers

indicating which

part of molecule

is being detected

followingfragmentation.

These markers

may be labeled

with masses.

MaxEnt 3TM for Sequencing



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

m/z0

100

%

Y;136.1

L

86.1

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

z5

644.4

z7

897.5757.9

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

mass0

100

%

y''11

y''10

1285.7

Y

136.1

L

86.1

y''9

1156.7

a1

197.1y''7

914.6265.1

b2

388.2y''4

505.3 643.3 781.4

y''8

1042.7

z10

1268.69

z11

1431.81672.9a11

1513.8

1655.8

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 lab

http://www.nhmfl.gov/science/cimar/icr/

Introduction to mass spectrometry at SciMedia.com

http://www.rmsb.u-bordeaux2.fr/rmsb/ms/IntroMS.html

The Thermo Finnigan homepage

http://www.thermo.com/eThermo/CDA/BU_Home/BU_Homepage/0,12482,113,00.html

The Micromass homepage, Mass Spec Back to Basics course

http://www.micromass.co.uk/basics/default.asp

Mass Spec Glossary

http://www.genomicglossaries.com/content/mass_spectrometry.asp

The I-mass homepage

http://www.i-mass.com/

I-mass tutorials

http://www.i-mass.com/guide/tutorial.html

American Society for Mass Spectrometry: What is Mass Spectrometry

http://www.asms.org/whatisms/

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Fundamentals of Mass Spec

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