Mass Spectroscopy

Mass Spectroscopy

Alireza Ghassempour (PhD)

Medicinal Plants and Drugs Research InstituteShahid Beheshti University

Evin, Tehran

Operational sequenceOperational sequence

introduce introduce samplesample ionizeionize

separate by separate by mass/chargemass/charge

detect ionsdetect ions

Sample introductionSample introduction

Ionization methodsIonization methods

EI – direct interaction of electrons with sampleEI – direct interaction of electrons with sample

CI – electrons ionize reagent gasesCI – electrons ionize reagent gases

DI – uses a pulse of energy to produce ionsDI – uses a pulse of energy to produce ions

SI – converts solvated molecules into ionsSI – converts solvated molecules into ions

electron ionizationelectron ionization

There will be different degrees of fragmentation There will be different degrees of fragmentation depending on the stability of the sample moleculedepending on the stability of the sample molecule

For a stable aromatic compound the primary peak is the parent ionFor a stable aromatic compound the primary peak is the parent ion

For a less stable cyclic compound fragmentation is predominantFor a less stable cyclic compound fragmentation is predominant

Chemical ionizationChemical ionization

Chemical ionization is a more controlled method Chemical ionization is a more controlled method of ionization than electron ionizationof ionization than electron ionization

In CI a neutral analyte (M) reacts with a reagent In CI a neutral analyte (M) reacts with a reagent ion that is generated by EI to form a variety of ion that is generated by EI to form a variety of molecular ionsmolecular ions

**

Desorption ionizationDesorption ionization

1.1. Energetic primary ions Energetic primary ions secondary ion mass spectrometry (SIMS)secondary ion mass spectrometry (SIMS)

2.2. Energetic atoms Energetic atoms fast atom bombardment (FAB)fast atom bombardment (FAB)

3.3. Nuclear fission fragments Nuclear fission fragments plasma desorption (PD)plasma desorption (PD)

4.4. Photons Photons laser desorption (LD)laser desorption (LD)

5.5. Very rapid heatingVery rapid heatingdesorption chemical ionization (DCI)desorption chemical ionization (DCI)

FAB ionization matricesFAB ionization matrices

Optimal matrix propertiesOptimal matrix propertiesStrongly absorbs the energy providedStrongly absorbs the energy provided

Contributes few ions to the spectrumContributes few ions to the spectrum

Interacts with the analyte to produce ionsInteracts with the analyte to produce ions

Effectively transfers energy to the analyte ionsEffectively transfers energy to the analyte ions

Diagram of an FAB gun. 1, Ionization of argon; the resultingions are accelerated and focused by the lenses 2. In 3, the argon ions exchange their charge with neutral atoms, thus becoming rapid neutral atoms. As the beam path passes between the electrodes 4, all ionic species are deflected. Only rapid neutral atoms reach the sample dissolved in a drop of glycerol, 5. The ions ejected from the drop are accelerated by the pusher, 6, and focused by the electrodes, 7, towards the analyser, 8.

Depending on the nature of the matrix we can obtain different molecular ionsDepending on the nature of the matrix we can obtain different molecular ions

protonated protonated speciesspecies

deprotonated deprotonated speciesspecies

Ag saltAg salt

Why are there two peaks?

107Ag and 109Ag

Spray ionization methodsSpray ionization methods

Spray ionization achieves the direct conversion of non-volatile, Spray ionization achieves the direct conversion of non-volatile, solvated molecules into gas phase ionssolvated molecules into gas phase ions

Electrospray (ESI)

Electrospray ionizationElectrospray ionization

ESI-MS of cytochrome c (mw = 12,360)

The isotope distribution also allows charge assignment, since each The isotope distribution also allows charge assignment, since each isotopic peak is separated from the next by 1/n where n is the chargeisotopic peak is separated from the next by 1/n where n is the charge

peak separation is 1/15peak separation is 1/15

Principles of MALDI

The sample is dispersed in a large excess of matrix material which will strongly absorb the incident light.

The matrix contains chromophore for the laser light and since the matrix is in a large molar excess it will absorb essentially all of the laser radiation

The matrix isolates sample molecules in a chemical environment which enhances the probability of ionization without fragmentation

Short pulses of laser light (UV, 337 nm) focused on to the sample spot cause the sample and matrix to volatilize

The ions formed are accelerated by a high voltage supply and then allowed to drift down a flight tube where they separate according to mass

Arrival at the end of the flight tube is detected and recorded by a high speed recording device

Matrix Assisted Laser Desorption

Matrices

Matrix

1,8,9-Trihydroxyanthracen(Dithranol) polymers

2,5-Dihydroxy­benzoic­acid(DHB)

proteins,­peptides,­polymers

-Cyano-4-hydroxycinnamic­acid

peptides,­(polymers)

4-Hydroxypicolinic­acid oligonucleotides

Trans-Indol-3-acrylacid(IAA)

polymers

OH OH OH

OH

OH

COOH

C CH

OH

CN COOH

N

OH

COOH

NH

COOH

Sample Preparation: Dried Droplet

solved­Matrix solved­sample

Mixing­and­Drying

Sample Preparation: Thin Layer

solved­Matrix

solved­sample

fastdrying

thin homogenuouslayer of crytslas

Drying

MALDI spectra of a monoclonal antibody (above) and of a polymer PMMA 7100 (below).

Secondary Ion Mass Spectrometry (SIMS)

Secondary ion generation

The sample is prepared in an ultra high vacuum.

A beam of primary ions or neutral particles impacts the surface with energies of 3-20 keV.

A primary ion or particle causes a collision cascade amongst surface atoms and between .1 and 10 atoms are usually ejected. This process is termed sputtering. The sputter yield depends on the nature of the analyte.

Static SIMS Low ion flux is used. This means a small amount of

primary ions is used to bombard the sample per area per unit time. Sputters away approximately only a tenth of an atomic monolayer.

Ar+, Xe+, Ar, and Xe are the commonly used particles present in the primary particle beam, which has a diameter of 2-3 mm.

The analysis typically requires more than 15 minutes. This technique generates mass spectra data well

suited for the detection of organic molecules.

Imaging SIMS The mass spectrometer is set

to only detect one mass. The particle beam traces a

raster pattern over the sample with a low ion flux beam, much like Static SIMS.

Typical beam particles consists of Ga+ or In+ and the beam diameter is approximately 100 nm.

The analysis takes usually less than 15 min. The intensity of the signal detected for the particular mass is

plotted against the location that generated this signal. Absolute quantity is difficult to measure, but for a relatively

homogeneous sample, the relative concentration differences are measurable and evident on an image.

Images or maps of both elements and organics can be generated.

Images created using the Imaging SIMS mode.

Scanning ion image of granite from the Isle of Skye.-University of Arizona SIMS 75 x 100 micrometers.

Mass Analyzers

Mass Analyzer divided into:

1. Scanning analysers transmit:1.1 only the ions of a given mass-to-charge

ratio to go through at a given time (magnetic, qudrupole)

1.2. allow the simultaneous transmission of all ions (ion trap, TOF)

2. ion beam versus ion trapping types,

3. continuous versus pulsed analysis,

4. low versus high kinetic energies

The five main characteristics for mass analyser:

1. The mass range limit (Th)

2. The analysis speed (u s−1)

3. The transmission (the ratio of the number of ions reaching the detector and the number of ions entering the mass analyser, a quadrupole MS used in SIM mode has a duty cycle of 100 % but a quadrupole MS scanning over 1000 amu, the duty cycle is

1/1000=0.1%. )

4. The mass accuracy (ppm)

5. The resolution.

Two peaks are considered to be resolved if the valley between them is equal to 10% of the weaker peak intensity when using magnetic or ion cyclotron resonance (ICR) instruments and 50% when using quadrupoles, ion trap, TOF, and so on.

R=m/∆m

Low resolution or high resolution is usually used to describe analysers with a resolving power that is less or greater than about 10 000 (FWHM), respectively

The first example is human insulin, a protein having the molecular formula C257H383N65O77S6.

The nominal mass of insulin is 5801 u using the integer mass of the most abundant isotope of each element, such as 12 u for carbon, 1u for hydrogen, 14 u for nitrogen, 16 u for oxygen and 32 u for sulfur.

Its monoisotopic mass of 5803.6375 u is calculated using the exact masses of the predominant isotope of each element such as C=12.0000 u, H=1.0079 u, N=14.0031 u, O=15.9949 u and S=31.9721 u.

Monoisotopic mass / Average Mass

Monoisotopic mass

Average mass

Magnetic-Sector

THEORY:

The ion source accelerates ions to a kinetic energy given by:

KE = ½ mv2 = qV

Where m is the mass of the ion, v is its velocity, q is the charge on the ion, and V is the applied voltage of the ion optics.

Magnetic-Sector

•The ions enter the flight tube and are deflected by the magnetic field, B.

•Only ions of mass-to-charge ratio that have equal centripetal and centrifugal forces pass through the flight tube:

mv2 /r = BqV, where r is the radius of curvature

Magnetic-Sector

mv2 /r = BqV

•By rearranging the equation and eliminating the velocity term using the previous equations, r = mv/qB = 1/B(2Vm/q)1/2

•Therefore, m/q = B2r2/(2V)

•This equation shows that the m/q ratio of the ions that reach the detector can be varied by changing either the magnetic field (B) or the applied voltage of the ion optics (V).

Basis of Quadrupole Mass Filter

consists of 4 parallel metal rods, or electrodes

opposite electrodes have potentials of the same sign

one set of opposite electrodes has applied potential of [U+Vcos(ωt)]

other set has potential of

- [U+Vcosωt] U= DC voltage, V=AC voltage,

ω= angular velocity of alternating voltage

The trajectory of an ion will be stable if the values of x and y never reach r0, thus if it never hits the rods. To obtain the values of either x or y during the time, these equations need to be integrated. The following equation was established in 1866 by the physicist Mathieu :

Mass analyzersMass analyzers

An ion trap is a device that uses an oscillating electric field to store ions. The ion trap works by using an RF quadrupolar field that traps ions in two or three dimensions (2D and 3D).

Ion Trap MS Ions are trapped by applying

rf frequencies on the ring electrode and endcaps

Then ions are scanned out of the trap by m/z as the base mass voltage is increased over time

This is accomplished by maintaining in the trap a pressure of helium gas which removes excess energy from the ions by collision.

• The Back Plate and Grid are used to accelerate the ions• The Ion source is used to ionize the Sample

• The Sample Inlet introduces the sample to the source• The vacuum is used to maintain a low pressure

• The Drift Region separates the ions according to their mass• The Detector outputs current as each ion strikes it

• The Oscilloscope displays the detector output

Time of Flight Schematic

Time-of-Flight Converted to Mass An accelerating potential (V) will give an ion of charge z an energy of zV. This can be

equated to the kinetic energy of motion and the mass (m) and the velocity (v) of the ion

zV = 1/2mv2

Since velocity is length (L) divided by time (t) then

m/Z = [2Vt2]/L2

V and L cannot be measured with sufficient accuracy but the equation can be rewritten

m/Z = B(t-A)2

where A and B are calibration constants that can be determined by calibrating to a known m/Z

Reflectron TOF-MS Improved mass resolution in MALDI TOF-MS has been obtained by the utilization of a single-stage or a dual-stage reflectron (RETOF-MS). The reflectron, located at the end of the flight tube, is used to compensate for the difference in flight times of the same m/z ions of slightly different kinetic energies by means of an ion reflector. This results in focusing the ion packets in space and time at the detector

A typical MALDI mass spectrum of substance P in CHCA (see Table 1) employing both linear and reflectron TOF-MS in the continuous ion extraction mode with a 500 MS/s transient digitizer is shown. The maximum mass resolution observed in the linear mass spectrum of substance P employing continuous ion extraction is about 600 which is typical for a peptide of this size. Only the average chemical mass can be determined from this mass spectrum. In the reflectron mass spectrum, the isotopic multiplet is well resolved producing a full width half maximum (FWHM) mass resolution of about 3400

FT-ICR-MS instrument general scheme

FTICR: New Dimensions of High Performance Mass Spectrometry

Ions­are­trapped­and­oscillate­with­low,­incoherent,­thermal­amplitudeExcitation­sweeps­resonant­ions­into­a­large,­coherent­cyclotron­orbit­Preamplifier­and­digitizer­pick­up­the­induced­potentials­on­the­cell.

FTICR: New Dimensions of High Performance Mass Spectrometry

The­frequency­of­the­cyclotron­gyration­of­an­ion­is­inversely­proportional­to­its­mass-to-charge­ratio­(m/q)­and­directly­proportional­to­the­strength­of­the­applied­magnetic­field­B.

B

v

BvqFL

+

Excitationelectrodes

Detection electrodes

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

+

-100

-80

-60

-40

-20

0

20

40

60

80

100

Inte

nsi

ty [

%]

Time

ExciteDetect77 - Frequency spectrumWideband (BW: 500.000k) accumulated 5 scans

Frequency [Hz] x1E350 60 70 80 90 100 110 120 130 140 150 160 170

Inte

ns

ity

[%

]

0

20

40

60

80

100

Frequency

Fourier Transform

Time

0

Inte

ns

ity

[%

]

m

qBf

20

MS spec with only one frequency

Fourier Transform

ExciteDetect77 - Magnitude spectrumWideband (BW: 500.000k) serial scan 21 of 50

874.87

874.94

875.01

875.08

875.15875.22

875.29875.36

875.43

875.50

875.58

875.65

875.72

Masses [m/z]874.8 874.9 875 875.1 875.2 875.3 875.4 875.5 875.6 875.7 875.8

Inte

ns

ity

[%

]

0

10

20

30

40

50

60

70

80

90

100

110

High Resolution of FTICR MS

Ubiquitin (14+)

Mass resolution: 170,000

(up to 5,000,000)

mass

Finnigan LTQ FT Ultra

51

Finnigan LTQ FT Ultra

52

Biological samplesCeribrospinal fluid (CSF), 1µL Injection volume200 nL/min, Nanospray, D:\Data\...\HUPO\CSF79_120-30_SIM_LC154 18.08.2005 06:06:46

RT: 0.00 - 150.00

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nc

e

95.17

56.02

80.5469.62104.4856.58

20.47 80.90141.4170.0750.39

86.3922.48

96.9290.53

38.31 143.1873.7426.79 110.7665.7435.99 114.5744.45 130.52

117.43 134.06123.0620.11 33.3518.4814.289.68

NL:2.70E8

TIC F: MS CSF79_120-30_SIM_LC154

CSF79_120-30_SIM_LC154 #7925 RT: 49.57 AV: 1 NL: 1.40E6T: FTMS + p NSI Full ms [ 300.00-2000.00]

400 600 800 1000 1200

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nc

e

660.91

499.75

551.09

451.27

825.89

702.55613.77

435.77330.46

850.90737.35936.72 1207.85319.86 1080.97

CSF79_120-30_SIM_LC154 #7929 RT: 49.60 AV: 1 NL: 2.01E5T: ITMS + c NSI d Full ms2 [email protected] [ 170.00-2000.00]

200 400 600 800 1000 1200 1400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nc

e

838.67

394.99

767.20

542.31743.35 1028.77458.38 1138.85337.49

876.52 1396.211214.29

MS Peptide mass MS/MS Peptide sequence

TIC Separation of peptides

660.91

m/z m/z

Retention Time, min

Finnigan LTQ FT Ultra – MS/MS

54

Biological samplesCeribrospinal fluid (CSF), 1µL Injection volume200 nL/min, Nanospray, D:\Data\...\HUPO\CSF79_120-30_SIM_LC154 18.08.2005 06:06:46

RT: 0.00 - 150.00

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nc

e

95.17

56.02

80.5469.62104.4856.58

20.47 80.90141.4170.0750.39

86.3922.48

96.9290.53

38.31 143.1873.7426.79 110.7665.7435.99 114.5744.45 130.52

117.43 134.06123.0620.11 33.3518.4814.289.68

NL:2.70E8

TIC F: MS CSF79_120-30_SIM_LC154

CSF79_120-30_SIM_LC154 #7925 RT: 49.57 AV: 1 NL: 1.40E6T: FTMS + p NSI Full ms [ 300.00-2000.00]

400 600 800 1000 1200

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nc

e

660.91

499.75

551.09

451.27

825.89

702.55613.77

435.77330.46

850.90737.35936.72 1207.85319.86 1080.97

CSF79_120-30_SIM_LC154 #7929 RT: 49.60 AV: 1 NL: 2.01E5T: ITMS + c NSI d Full ms2 [email protected] [ 170.00-2000.00]

200 400 600 800 1000 1200 1400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lati

ve

Ab

un

da

nc

e

838.67

394.99

767.20

542.31743.35 1028.77458.38 1138.85337.49

876.52 1396.211214.29

MS Peptide mass MS/MS Peptide sequence

TIC Separation of peptides

660.91

m/z m/z

Retention Time, min

LC-MS/MS

56

Orbitrap- a new type of FTMS

Click to edit Master title style

58

Principle of Trapping in the Orbitrap

Orbital trapsKingdon (1923)

The Orbitrap is an ion trap – but there are no RF or magnet fields!

• Moving ions are trapped around an electrode

- Electrostatic attraction is compensated by centrifugal force arising from the initial tangential velocity. Analogon: a satellite is “trapped” around the Earth; a satellite is “trapped” around the Earth; gravitational attraction is compensated by centrifugal gravitational attraction is compensated by centrifugal force arising from initial tangential velocity.force arising from initial tangential velocity.

• Potential barriers created by end-electrodes confine the ions axially

• The frequencies of oscillations (especially the axial ones) could be controlled by shaping the electrodes appropriately

• Thus we arrive at …

LTQ Orbitrap™ Hybrid Mass SpectrometerLaunched in Summer 2005

API Ion source Linear Ion Trap C-Trap

Orbitrap

Finnigan LTQ™ Linear Ion Trap

Differential pumping

Differential pumping

Inventor: Dr. Alexander Makarov, Thermo Fisher Scientific (Bremen, Germany)

LTQ Orbitrap Operation Principle

1. Ions are stored in the Linear Trap2. …. are axially ejected3. …. and trapped in the C-trap4. …. they are squeezed into a small cloud and injected into the Orbitrap5. …. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation

The oscillating ions induce an image current into the two outer halves of the Orbitrap, which can be detected using a differential amplifier

Ions of only one mass generate a sine wave signal

The axial oscillation frequency follows the formula Where = oscillation frequency

k = instrumental constant m/z = …. well, we have seen this before

zm

k

/

Frequencies and Masses

Many ions in the Orbitrap generate a complex signal whose frequencies are determined using a Fourier Transformation

What‘s the size of the Orbitrap?

LTQ Orbitrap™ Hybrid Mass Spectrometer

Tandem mass spectrometersTandem mass spectrometers

Uses of tandem mass spectrometry:Uses of tandem mass spectrometry:

(1)(1) Characterize individual compounds in complex mixturesCharacterize individual compounds in complex mixtures

(2)(2) Completely identify the molecular structure of a compoundCompletely identify the molecular structure of a compound

To accomplish either of these goals mass analysis To accomplish either of these goals mass analysis must be carried out twice in a tandem instrumentmust be carried out twice in a tandem instrument

This can be achieved either by separating the This can be achieved either by separating the mass analysis operations in space or in timemass analysis operations in space or in time

Tandem mass spectrometersTandem mass spectrometers

Information obtained from MS experiments

Chemical Ionization(CI)

Electron impact Ionization(EI)

GC-MS LC-MS

-TOF Product (185.0): 1.750 to 3.300 min from pinic_P185.wiffa=3.56215314471457500e-004, t0=4.61223072761931690e+001

Max. 46.2 counts.

115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195m/z, amu

0

5

10

15

20

25

30

35

40

45

Inte

ns

ity, c

ou

nts

141.09268

185.08193123.08251 167.07195

-TOF Product (185.1): 1.267 to 2.317 min from Sample 5 (ESI-: pinic P185 CE=-6, CAD=2) of 0...a=3.56228408459780370e-004, t0=4.72207389233553840e+001

Max. 170.6 counts.

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190m/z, amu

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

Inte

ns

ity, c

ou

nts

185

COOH

COOH

MS

MS/MS

Molecular weight

Structural information

Separation in space can be achieved by coupling two mass analyzersSeparation in space can be achieved by coupling two mass analyzers

For example, a sector magnet (MS 1) can be coupled to a For example, a sector magnet (MS 1) can be coupled to a quadrupole mass filter (MS 2)quadrupole mass filter (MS 2)

A parent ion is selected by the sector magnet and A parent ion is selected by the sector magnet and separated from the other ions in the sampleseparated from the other ions in the sample

Each selected ion is activated by a collision processEach selected ion is activated by a collision process

The resulting set of product ions are analyzed by the The resulting set of product ions are analyzed by the quadrupole mass filterquadrupole mass filter

Tandem mass spectrometersTandem mass spectrometersThe most common tandem mass spectrometer The most common tandem mass spectrometer

is a triple quadrupole mass spectrometeris a triple quadrupole mass spectrometer

Mass Spec Ion Detectors

Faraday Cup Electron Multiplier and Channel Electron

Multiplier Microchannel Plate Daly Detector (Scintillation Counter or

Photomultiplier)

Electron Multipliers

Continuous Electron Multiplier

Photomultiplier (Daly detector)

Photomultiplier

Also called daly detector or scintillation counter Metal dynode emits secondary electrons Secondary electrons hit phosphorus screen and

trigger photon emission; photon abundance measured by photomultiplier

Advantage: keep detector in vacuum -- no contamination = low noise and long lifetime

Disadvantage: cannot be exposed to light

Data Collection

Typically, the computer controls: Scanning of mass spec Data acquisition Data processing Interpretation of data (generation of spectra;

possible comparison to spectra in database)

Sequencing Using MALDIWhen the molecular mass of a peptide is known, for example Gly2Tyr = 296, we may use a digested, or fragmented effect of MALDI to learn the sequence of the peptide. Backbone (peptide bond) cleavages in the mass spectrometer generate two types of ions. Acylium ions are produced when the charge is retained on the N-terminal side of the peptide, while protonated peptides are produced when the charge is retained on the C-terminal side of the peptide. These ion fragments are known as bn and yn fragments respectively. In straightforward cases, all possible fragments are produced using prior peptide digestion. This means that for a given peptide sequence, two unique sets of fragment ions are produced (the bn and yn sets). In the following table, the “predicted” fragments for a given peptide are listed. Comparison of these predicted fragments with experimentally observed peptide fragments allows for a given peptide mass to be assigned to a unique peptide sequence.

Formulas for prediction of mass spectrometer fragments during the analysis of peptides.

Two series (bn and yn) are produced, depending on the peptide end on which cleavage occurs

b1 Mresidue + H y1 Mresidue + 19

b2 Mresidue + b1 y2 Mresidue + y1

bn-1 MH+ - 18 - Mresidue Yn-1 MH+ - Mresidue

ExampleConsider the peptide GlyTyr. Two possible sequences are possible:

H-Gly-Tyr-OH H-Tyr-Gly-OH

The predicted fragments for these two sequences would be:

H-Tyr-Gly-OH H-Gly-Tyr-OH

b1 = 164 b1 = 58

b2 = 221 b2 = 221

y1 = 76 y1 = 182

y2 = 239 y2 = 239

Suppose experimentally, we detected fragments at mass 164, 221 but none at 239. This missing mass is sufficient to identify the sequence as H-Tyr-Gly-OH.

Peptide Fingerprinting

MALDI may be used to determine an exact “peptide fingerprint” of a section of a protein that carries a “disease” or differs from the normal protein in some way.

C-1

C-2

C -3

Tox-1

Tox-2

Tox-3

Standard Control 2D Gel

Results: 196 features detected; 40 ( ) at 2-fold change.

Digitized Image

Digitized PDQuest image following matchset comparison, which allows comparison of all control versus all toxicant images. Each black spot represents a protein; spots circled in yellow indicate prot. eins with at least a 2-fold change based on spot intensity or statistical test

Image Analysis for Differential Protein Expression

Protein Identification by MALDI-MS• Differentially expressed proteins identified by image analysis are excised from 2D gels and trypsin digested. The resulting peptide fragments are analyzed on a MALDI mass spectrometer (MS).

• The MALDI spectra displays a “peptide fingerprint” of the protein using corresponding peptide masses.

Database Searching using MALDI-MS Ions for Protein Identification

• Proteins are identified by entering the masses (ions from MALDI spectrum) of the peptides into a peptide mapping database such as ProFound.

• Search parameters are refined by including experimental mass and isoelectric point (pI) determined by 2D PAGE, as well as by taxonomic category and any modifications.

http://prowl.rockefeller.edu/cgi-bin/ProFound