MASS SPECTROMETRY

MALDI-TOF AND ESI-MS

Topics
Principle of Mass Spectrometry MALDI-TOF Determination of Mw of Proteins Structural Information by MS: Primary Sequence of a Protein

A. Principles
Ionization: by Ion Source Production of ions in vacuum (~10-5 Pa or 9.8 10-11 atm) To prevent reaction between ions and air molecules Separation of Ions: in Mass Analyzer Separation of ions according to mass-tocharge ratio (m/z) Detection of ions Storage of Data Analysis

MALDI (1988): Soft Ionization Method

MALDI makes it possible to introduce large biomolecules into vaccum without fragmentation Provides accurate molecular mass. Relative error of 0.1-0.01% and even smaller are possible Extremely sensitive (down to femtomolar quantities) Broad mass range High resolution Relatively tolerant of buffers and salts Simple mixtures can be analyzed Data collected can be submitted automatically for database search.

B-1 Ionization
Low concentration analyte is dispersed in a solid or liquid matrix and deposited on a metal plate Typical analyte to matrix ratios: 1:103 to 1:105. Plate is placed in vacuum chamber where a laser beam is focused onto the sample Matrix must strongly absorb the laser radiation Matrix and analyte are desorbed and ionized Ions are accelerated towards the drift tube (TOF mass analyser)

Proposed Mechanism of Ionization

Absorption of laser beam energy by matrix molecules Transfer of energy from matrix molecules to analyte molecules Desorption of analyte and matrix molecules Analyte molecules are desorbed as neutral molecules Analyte is ionized by proton-transfer with protonated matrix ions

Matrices

Lasers
Nitrogen: 337 nm Nd-YAG: Neodynium-Yttrium Aluminium Garnet: 266 and 355 nm Pulse length : 1-5 nanoseconds

Mass Analysis in TOF Analyzer

The DRIFT TUBE

Theoretically, MALDI TOF is limitless in its ability to measure m/z Practically: can accurately measure masses up to ~300 kDa

(1) E kin =

m : mass v : velocity z : ch arg e e : elementary ch arg e V : voltage L ( 2) v = tF L : length of drift tube m 2e.V 2 (3) = 2 t F z L m (4)t F = L 2 zeV

1 2 mv = z.e.V 2

Calibration: Measure time of flight of standards of known m/z to obtain calibration constants Measure t for unknown Calculate mass

Calibration and Determination of Mass

(3) m 2e.V 2 2 = 2 t F = Const.t F z L m (5)t F = Const z (6)t F = Const a + Const b Flex control : t F = C0 + Solve for m z m m + Cont c z z 1012 m m . + C2 . C1 z z

Constant a accounts for uncertainties in the start time Variations in Constant b account differences in the energy of the ions due mainly to the topology of the matrix-preparation and to a lesser extent to the geometric variations of the target Constant c: correction for higher order errors

The problem: Peaks are inherently broad in MALDI-TOF spectra (poor mass resolution). The cause: Ions of the same mass coming from the target have different speeds. This is due to uneven energy distribution when the ions are formed by the laser pulse.
Sample + matrix on target Ions of same mass, different velocities + + +

Can we compensate for the initial energy spread of ions of the same mass to produce narrower peaks?

Delayed Extraction Reflector TOF Mass Analyzer

Delayed Extraction (DE) improves performance

0 V. + + + 0 V. Ions of same mass, different velocities

Step 1: No applied electric field. Ions spread out. 20 kV. + + +

0 V. Step 2: Field applied. Slow ions accelerated more than fast ones. + + + 0 V.

20 kV. Step 3: Slow ions catch up with faster ones.

What is a reflector TOF analyzer?

A single stage gridded ion mirror that subjects the ions to a uniform repulsive electric field to reflect them. Detector

Ion Source

Reflector (Ion Mirror)

The reflector or ion mirror compensates for the initial energy spread of ions of the same mass coming from the ion source, and improves resolution.

A reflector focuses ions to give better mass resolution

+ +

0 V.

+20 kV

Resolution & mass accuracy on mellitin

8000

15 ppm error

Resolution = 18100

6000

Resolution = 14200
Counts
4000

24 ppm error
Resolution = 4500

2000

55 ppm error
0 2840 2845 2850 2855

Mass (m/z)

26 amino acid peptide: 50 % of dry weight of bee venom

Isotope effect on MALDI spectrum

A Element H C N O F P S Cl

A+1

A+2

Element Type

mass %abund mass %abund mass %abund 1 12 14 16 19 31 28 35 100 100 100 100 100 100 100 100 2 13 15 17 0.015 1.1 0.37 0.04 A+1 A+1 A+1 0.2 A+2 A A 3.4 A+2 32.5 A+2

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33

0.8

34 37

Post Source Decay (PSD) (MS/MS)

1. PSD refers to a method of detecting and measuring the masses of fragment ions that are formed from a selected precursor ion. 2. Fragment ions are mainly formed by unimolecular decomposition after the precursor ions are fully accelerated (after they exit the sourcehence post-source decay) 3. Fragment ions are separated and detected in the reflector.

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Decomposition occurs in the flight tube

Laser

Reflector detector

Linear detector

Source

Decay can occur at any point along here

Reflector

Internal energy of precursor ions

Only a small fraction of the precursor ions have enough energy to fragment during their lifetimes.

No of ions

Internal energy
For peptides the efficiency of PSD fragmentation is amino acid composition and sequence dependent.

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Increasing PSD Fragmentation

There are two ways to increase the amount of fragmentation: both act to increase the precursor ions internal energy. Use higher laser intensity Use a collision cell

PSD fragment ion velocities are the same as their precursors

+ + + All three of these species travel at the same velocity in the flight tube until they reach the reflector.

Why? Velocity is determined by initial acceleration. Initial energy = 20 keV. Bond energies = ~ 10 eV, so breaking a bond has a very minor effect on velocities.

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Timed Ion Selector (TIS)/PreCursor Ion Selector(PCIS)

The TIS is a Bradbury-Neilson gate, which is a type of velocity selector. It allows only selected precursor ions and their fragments to pass through to the reflector. Gate closed: alternating potentials on wires Ions Gate open: wires at ground potential
+

Timed Ion Selector operation

TIS off
Gate open

TIS on
Gate closed

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Effect of the timed ion selector

Before fragmentation
The intact molecular ion has translational kinetic energy equal to:

KE = 1/2 Mv2
where:

KE = kinetic energy (= z eV) M = mass v = velocity

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Post source fragmentation

The translational kinetic energy of a fragment ion is

m KE m = KE M M
where KEM = precursor kinetic energy KEm = fragment kinetic energy M = precursor mass m = fragment mass

Precusor and PSD fragment ions take different paths in the normal reflector
Reflector detector
Intact precursor ion

+ +
Fragment ion formed by PSD 0 V.

Reflector

+20 kV

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How are PSD fragment ions that are traveling at the same speed as the precursor ion but contain reduced kinetic energy made to arrive at the detector so that they are focused?

By varying the steepness of the voltage gradient in the reflector across the fragment ion mass range.

PSD mirror ratio setting

Consider an ion (MH+) that can decompose into two fragments, A and B. Either of the following reactions can occur: MH+ MH+ AH+ + B A + BH+

Assume MH+ = 1,000 Da, AH+ = 700 Da, and BH+ = 300 Da

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At mirror ratio = 1.00

AH+ BH+ MH+

MH+ ( 1,000) correctly focused AH+ (700) Poorly focused BH+ (300) Poorly focused

At mirror ratio = 0.7

BH+ AH+ MH+

MH+ ( 1,000) not focused AH+ (700) correctly focused BH+ (300) Poorly focused

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At mirror ratio = 0.3

BH+

AH+ & MH+

MH+ ( 1,000) not focused AH+ (700) not focused BH+ (300) correctly focused

A PSD spectrum is taken in stitches

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu formed by the action of renin on angiotensinogen. Renin is produced in the kidneys in response to both decreased intra-renal blood pressure

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B-4 Resolution
m m = m m2 m1

Rs =

m : full width at half max imum( FWHM )

Typically: ~15,000 and higher

B.5 Applications of MALDI

Analysis of Proteins and Peptides MW Structural information Post-translational processes Sequencing Identification of a protein based on analysis of a digest finger print using proteins digest finger prints data base Analysis of Mixtures of Proteins and Peptides
Elminates need for separation

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Intens. [a.u.]

Intens. [a.u.]

Intens. [a.u.]

Intens. [a.u.]

D
0 1 2 0 1 1 x10 4 3 0 x10 4

x10 4 6

x10 4

0 00

3152.1 3392.7 3604.6 3604.5 3872.4 3702.7

4000

3932.3 4259.8 4493.6 4697.9 4697.8

3151.5

3214.1 3422.4 3839.3 4140.9

4303.8 4534.4 4673.7 4795.1 5042.5 5462.4 5691.8 5426.9 5565.2 5944.8 5750.3 5959.3 6099.4 6305.7 6250.9 6261.4

5000

5052.1 5462.4 5692.7

6000

6099.9 6481.8

6657.8 6860.6
7000

Courtesy of Prof. Ouellette

6790.3 7088.0 7333.8 7748.5
8000

6851.0 7333.1 7747.9 7323.4 7728.2 7249.9

8343.8

8343.5

8229.1 8484.2

8080.0 8289.5 8726.7

9000 10000 11000 12000

8922.5 9186.1 9395.3 9624.1 9999.8

9012.9

8954.5 9351.7

9075.2 9342.7

9999.2 10311.9 10645.9 10926.0 11135.1 11386.5 11754.5

10058.9 10311.5 10648.9 10454.8 10723.9 10925.4 11135.3 11385.9 11754.3 11134.3

10090.8 10397.1 10859.8

Whole Cell Preparations MALDI-TOF Spectra

11507.2 12212.3 12255.8 12396.2 m/z 12506.0 12529.5

070606\AP_070606_L21_207\1SLin

070606\AP_070606_L14_106\1SLin

070306\ap_070306_K15_102\1SLin, Baseline subt.

Mass Spec Data\070606 - comparison\1SLin, Baseline subt.

Fig. 8. MALDI-TOF mass spectra of whole cell preparations. A. isolate 106, B. isolate 207, C. Isolate 102, D. isolate 104. Cells were prepared for mass spectrometry using a thin smear of cells on the target, and saturated alpha-cyano4-hydroxycinnamic acid in 50% acetonitrile/ 1.0% TFA was added. 104 102 207

Isolate 106

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