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Home , Chemical ionization, Electrospray ionization, Ion source, Ion trap, Mass (mass spectrometry), Mass spectrum

Ion Sources and Analyzers in Characterization

Principles of MS and MS/MS

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

Time of Flight Mass Filter Quadrupole Trap Fourier Transform Ion Cyclotron

Lenses and prisms focus and refract light.

Analogous systems can focus and deflect in a .

1. Get into the & ionize them. 2. Give the ions a defined or velocity. 3. Separate or sort the ions on the basis of that defined property. 4. Detect the ions & assign their . A very simple mass of Dioxide

Copyright ASMS: http://www.asms.org/whatisms/p5.html 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 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 M1 M2 dM Resolution is often defined as M/dM.

FWHM 25% valley “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 = More Complex 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. Analyzer Resolution: Average vs. Monoisotopic Masses

Average mass: The mass of an ion for a given empirical formula calculated using the relative average 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. (MS/MS)

Mass

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

Tandem Mass Spectrometer Tandem instruments have multiple mass analyzers. Mass Analyzers

Magnetic Sector and Double Focusing Instruments

Quadrupole Mass Filters

Quadrupole Ion Traps

Fourier Transform Ion Cyclotron Resonance

Time of Flight 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. The mass range is scanned as the 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 and hybrid Q-q-TOF for biopolymer analysis. MS/MS in a Triple Quadrupole (Q-q-Q) Mass Spectrometer Mass Analyzers: The

Facile MSn High resolution over narrow ranges Extremely Sensitive Fast Scanning Small Inexpensive Mass Analyzers: Fourier Transform Ion Cyclotron Resonance

Ions in a 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

Mass Analyzers: Time of Flight (TOF)

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

Linear TOF

Reflectron TOF Ion Sources

Gas Phase Ionization: Impact (EI) (CI)

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

Spray Ionization: (TSP) Atmospheric Pressure Chemical Ionization (APCI) Electrospray (atmospheric pressure ionization) (ESI, API) The 2002

John B. Fenn for MS

Koichi Tanaka ionization for MS

Kurt Wuthrich NMR for protein structures

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

Analyte: 10 – 1000 fmol 1 – 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 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 Energy zeV = ½ mv2 v = (2zeV/m)½ Ion Sources: Electrospray Very gentle and efficient way of getting gas phase ions from . A fine spray of charged droplets is generated in an . 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 Electrospray of Myoglobin

m1 = (M+n)/n m2 = (M+n+1)/(n+1)

+21 +12

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

Using adjacent pairs of ions, the of the myoglobin can be calculated very accurately. Tandem Mass Spectrometry (MS/MS) is the Method of Choice for Sequence Analysis of Peptides

Speed Sensitivity Tolerance for Amino-terminal Blocking Groups High Specificity for Protein Identification Tandem Mass Spectrometry (MS/MS)

Mass Spectrometer

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

Tandem Mass Spectrometer Tandem instruments have multiple mass analyzers. Tandem Mass Spectrometry : Product Ion Scan

1. “Parent” Ions are selected and isolated 2. Collision-Induced-Dissociation Results in fragmentation 3. “Daughter” Ions are characterized with the second mass analyzer Q1 Q2 Q3 MASS FILTER RF ONLY MASS FILTER

PRECURSOR ION NEUTRAL GAS PRODUCT ION SELECTION COLLISIONS DETECTION DETECTOR Tandem Mass Spectrometry: Precursor Ion Scan

1. “Product” Ion is selected and Q3 is parked 2. Q1 is scanned normally 3. Only precursors which fragment to produce selected product ion are detected. Q1 Q2 Q3 MASS FILTER RF ONLY MASS FILTER

PRECURSOR ION NEUTRAL GAS PRODUCT ION SELECTION COLLISIONS DETECTION ION SOURCE DETECTOR Tandem Mass Spectrometry: Neutral Loss Scan 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 fragmentation 4. Daughter” Ions are detected only when the specified loss occurs in Q2, indicating the presence of the moiety of interest. Q1 Q2 Q3 MASS FILTER RF ONLY MASS FILTER

PRECURSOR ION NEUTRAL GAS PRODUCT ION SELECTION COLLISIONS DETECTION ION SOURCE DETECTOR 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

Another way to 532 669 784 label an MS/MS spectrum is to draw lines through the structure, with 400 pointers indicating which part of is being detected following fragmentation. These markers may be labeled with masses.

Micromass “Back to Basics” http://www.micromass.co.uk/basics/index.html MaxEnt-3TM for Sequencing

y''11 1448.8 100 y''10 TM MaxEnt-3 1285.7

Y y''9 % 136.1 1156.7

a1 197.1 z11 L y''7 z10 a11 1672.9 y''8 1431.8 86.1 1268.69 1513.8 b2 y''4 914.6 1042.7 265.1 388.2 1655.8 505.3 643.3 781.4 mass 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

Y;136.1 100 y''10 2+ y''11 2+ 643.4 724.9

643.9 y''9 2+ a1 578.8 197.1 %

L z5 86.1 558.3 644.4 y''7 2+ z7 457.8 Raw data 293.1 897.5 757.9 m/z 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 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/