Mass Spectrometry i Post

8/13/2019 Mass Spectrometry i Post

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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


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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 airmolecules

Separation of Ions: in Mass Analyzer

Separation of ions according to mass-to-charge ratio (m/z)

Detection of ions

Storage of Data Analysis

--


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MALDI (1988): Soft Ionization Method

MALDI makes it possible to introduce large biomolecules intovaccum 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 databasesearch.

B-1 Ionization

Low concentration analyte isdispersed in a solid or liquidmatrix and deposited on a metalplate

Typical analyte to matrix ratios:1:103 to 1:105.

Plate is placed in vacuumchamber where a laser beam isfocused onto the sample

Matrix must strongly absorb thelaser radiation

Matrix and analyte are desorbedand ionized

Ions are accelerated towards thedrift tube (TOF mass analyser)


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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


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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


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zeV

mLt

t

L

Ve

z

mtubedriftoflengthL

t

Lv

voltageVechelementarye

echzvelocityvmassm

VezmvE

F

F

F

kin

2)4(

.2)3(

:

)2(

:arg:

arg:::

..2

1)1(

2

2

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

z

mforSolve

z

mC

z

m

CCtcontrolFlex

z

mCont

z

mConstConstt

z

mConstt

tConsttL

Ve

z

m

F

cbaF

F

FF

++=

++=

=

==

..10

:

)6(

)5(

..2

)3(

21

12

0

22

2

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


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The problem: Peaks are inherently broad in MALDI-TOF

spectra (poor mass resolution).

++

+

Sample + matrix on target

Ions of same mass, different velocities

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.

Can we compensate for the initial energy spread

of ions of the same mass to produce narrower

peaks?

Delayed Extraction

Reflector TOF Mass Analyzer


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Step 1: No applied electric field. Ions spread out.

+

+ +

Ions of same mass, different

velocities

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

0 V.

0 V.

+

+ +

Step 3: Slow ions catch up with faster ones.

20 kV.

20 kV.

0 V.

0 V.+

++

Delayed Extraction (DE) improves performance

Detector

Ion Source

What is a reflector TOF analyzer?

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 single stage gridded ion mirror that subjects the ions to a uniform repulsive electric

field to reflect them.


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0 V. +20 kV

A reflector focuses ions to give better mass

resolution

+

+

Resolution & mass accuracy on mellitin

0

2000

4000

6000

8000

Counts

2840 2845 2850 2855

Mass (m/z)

Resolution = 14200

Resolution = 4500

Resolution = 1810015 ppm error

24 ppm error

55 ppm

error

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


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Isotope effect on MALDI spectrum

A A+1 A+2 Element

Type

Element mass %abund mass %abund mass %abund

H 1 100 2 0.015 A+1

C 12 100 13 1.1 A+1

N 14 100 15 0.37 A+1O 16 100 17 0.04 18 0.2 A+2

F 19 100 A

P 31 100 A

S 28 100 33 0.8 34 3.4 A+2

Cl 35 100 37 32.5 A+2

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.

Post Source Decay

(PSD) (MS/MS)


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Laser

ReflectorSource

Linear

detectorReflector

detector

Decay can

occur at anypoint along here

Decomposition occurs in the flight tube

No of

ions

Internal energy

Only a small fraction of the precursor ions have enough

energy to fragment during their lifetimes.

Internal energy of precursor ions

For peptides the efficiency of PSD fragmentation is amino acid composition and

sequence dependent.


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There are two ways to increase the amount of

fragmentation: both act to increase the precursor

ions internal energy.

Use higher laser intensity

Use acollision cell

Increasing PSD Fragmentation

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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Effect of the timed ion selector

The intact molecular ion has translational kinetic energy equal

to:

KE = 1/2 Mv2

where:

KE = kinetic energy (= z eV)

M = mass

v = velocity

Before fragmentation


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MH+

BH+

AH+

MH+ ( 1,000) correctly focused

AH+ (700) Poorly focused

BH+ (300) Poorly focused

At mirror ratio = 1.00

MH+

BH+


MH+ ( 1,000) not focused

AH+ (700) correctly focused

BH+ (300) Poorly focused

AH+


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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

)(max:

12

FWHMimumhalfatwidthfullm

mm

m

m

mRs

=

=

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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00 4000 5000 6000 7000 8000 9000 10000 11000 12000

m/z

A

B

C

D

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-cyano-

4-hydroxycinnamic acid in 50% acetonitrile/ 1.0% TFA was added.

Isolate

106

207

102

104

Courtesy of Prof. Ouellette

Whole Cell Preparations MALDI-TOF Spectra

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