Ch 13. Basics of Mass Sppy()ectrometry (I) : Principles ...chem.yonsei.ac.kr/~mhmoon/pdf/advanced/Ch13-14_com.pdf · Basics of Mass Sppy()ectrometry (I): Principles & Ionization Sources

Ch 13. Basics of Mass Spectrometry (I)p y ( ): Principles & Ionization Sources

Why should you be interested in mass spectrometry (MS) ?

- to identify unknown compounds

- to quantify known materialsto quantify known materials

- to elucidate the structural and chemical properties

of moleculesof molecules

- detection of compounds at 10-12g, 10-15 mole for a

d f 100 d ltcompound of mass 100 dalton

1

1. Where are MS used?

• Biotechnology: analysis of proteins, peptides, oligonucleotides

• Pharmaceutical Analysis: d di bi t i l h i t h ki tidrugs discovery, combinatorial chemistry, pharmokinetics, drug metabolism

• Clinical Examination:Clinical Examination:neonatal screening, haemoglobin analysis, drug testing

• Environmental Analysis :Environmental Analysis : water, food, air quality (PCBs etc)

• Geological Analysis : g yoil composition

2

1.1 Instrumental Design of Mass Spectrometer

Ionization/ desorption Mass Sorting Detection

Analyzer Ion Detection

Source

+

DetectForm ions Sort Ions by Weight (m/z) Detect ions

Form ions

(charged molecules)

Sort Ions by Weight (m/z)

I l

10010010010075757575

Inlet

1330133013301330 1340134013401340 1350135013501350

50505050252525250000

• Solid• Liquid• Vapor

Data AnalysisSample IntroductionMethod to vaporize

sample

Mass Spectrum

3

Turbo pumpsDiffusion PumpsRough pumps Rotary pumps

High Vacuum SystemHigh Vacuum System

Inlet Ionsource

Mass Filter

Detector DataSystemsource Filter System

HPLC Electrospray TOF MicrochannelHPLCSample plateGCSolid probeDCI

ElectrosprayMALDIFABEI

TOFQuadrupoleIon TrapMagnetic Sector

Microchannel Plate (MCP)

Electron Multiplier

DCI CI FT-ICR

4

1.2 Vacuum TechnologyPressure Gauges

G P R T i l U

gy

Gauge Pressure Range Typical Use(torr)

Manometer 760-1 systems near atmThermocouple gauge 1 - 10-3 monitoring mechanical pumpsIonization gauge 10-3 - 10-11 high-vacuum systems

Vacuum Pumps

Pump Lowest Attainable Pressure Typical UsePump Lowest Attainable Pressure Typical UseMechanical pump 10-2 - 10-3 roughing or backing pumpDiffusion pump 10-6 vacuum linesTurbomolecular pump 10-9 high vacuum systemsTurbomolecular pump 10 9 high-vacuum systemsCryopump < 10-10 ultrahigh-vacuum sys.

5

1 3 Reason for Vacuum in MS 10-6 to mid 10-5 Torr1.3 Reason for Vacuum in MS 10-6 to mid 10-5 Torr

• Increase sensitivity

• Avoid ion-molecule reactions

• Collision free ion trajectories

• Increase filament lifetime

• Avoid electric dischargeAvoid electric discharge

• Avoid background interference

6

2 Sample Introduction Techniques2. Sample Introduction Techniques

• Direct Probe/Metal TargetsDirect Probe/Metal TargetsSample put onto the end of long probe and inserted into the MSSample spotted with matrix onto a metal plate

• Gas chromatography: EI, CISample must be volatile thermally stable- Sample must be volatile, thermally stable

• Liquid chromatography: FAB, APCI, ESI Widely used in pharmaceutical industryLC/MS applicable to thermally labile, high MW compoundsLC/MS suitable for proteins & peptidesO li t h iOn-line technique

7

3. Ionization Methods3. Ionization Methods

I i iIonization Techniques

ElectronImpact(EI)

ChemicalIonization(CI)

ESIFast AtomBombardment(FAB)

MALDI

Soft IonizationIntact

Hard IonizationFragments

(EI) (CI) (FAB)

IntactFragments

8

3.1 Electron Ionization (EI)3.1 Electron Ionization (EI)

M + e- (70 eV) M+ (5 eV) + 2e- (65 eV)

Excess E (65eV) in the molecule leads to some degree of fragmentationExcess E (65eV) in the molecule leads to some degree of fragmentation

M+ : molecular ions + fragment ions + neutral fragments

M + e- : M- (100 times less efficient)

•Energetic process. A heated filament emits electrons which are accelerated by a potential difference of usually 70eV into the sample chamber.

•Ionization of the sample occurs by removal of an electron from the molecule thus

9

o at o o t e sa p e occu s by e ova o a e ect o o t e o ecu e t usgenerating a positively charged ion with one unpaired electron.

1) Schematics of EI1) Schematics of EI

N M M+ 2N M + e- M+. + 2e-

Fragmentation

M+. A+ B+

A+

M+.

B+

10

2) Characteristics of EI 2) Characteristics of EI

Molecular ionMolecular ion eABeAB 2

))

Fragment ionFragment ion eBAeAB 2• Standard ionization methods in MS• Standard ionization methods in MS

• Vaporized sample bombards with high energy electrons (~ 70 eV)

• “Hard” ionization method leads to significant fragmentationg g

• Ionization is efficient but non-selective

• Widely used technique when coupled to GC

• Suitable for volatile organic compounds

eg. hydrocarbons, oils, flavors, fragrances

• Not really coupled to LC today• Not really coupled to LC today

• Produces M+. radical cation giving molecular weight

• Produces abundant fragment ions

11

g

• Library searchable spectra

EI Spectra

12

13

3.2 Chemical Ionization (CI)

• Positive Ion Chemical Ionization (PCI or CI)Positive Ion Chemical Ionization (PCI or CI)

• Negative Ion Chemical Ionization (NCI,NICI or ECNI)

1. Instrumentation for CI

2. Selection of Reagent Gas

3. Analytical Application y pp

14

1) EI – CI Comparison) p

EI CISource pressure

10-6 torr 0.2 –2 torr

Mean free path

-200 mm -2 x 10-4 mm

High Energy process Low Energy process

( th l )(possess thermal energy)

Odd E- ions formed Even E- ions formed

15

2) Advantages and Disadvantages of CI2) Advantages and Disadvantages of CI

Advantages of Chemical ionization:g1. Large (M+1)+ ion identifies molecular weight (M)

2. Sensitivity is enhanced by

- simple fragmentation (fewer peaks of higher abundance)

- direct GC/MS interface

3. CI spectra complement EI spectra

Disadvantages of Chemical ionization :Disadvantages of Chemical ionization :1. Simple fragmentation gives little structural information

2 E i t i ti2. Easy ion source contamination

16

3) What happens in the CI source ? ) pp

•Reactant ions are formed via EI/CI:

CH4 + e- CH4+ + 2e-

CH4 + e- CH3+ + H• + 2e-CH4 e CH3 H 2e

CH4 + e- CH2+ + H2 + 2e-

CH4+ + CH4 CH5

+ + CH3 •

CH3+ + CH4 C2H5

+ + H23 4 2 5 2

CH2+ + 2 CH4 C3H5

+ + 2H2 +H•

At 1 t th j i th tAt 1 torr, the major ions are those at

17amu (CH5+), 29amu (C2H5

+), 41amu (C3H5+ )

17

S l I i ti•Sample Ionization:

1. Proton transfer: higher proton affinity than that of reagent

CH5+ + RH CH4 + RH2

+ (M+H)+

C H + + RH C H + RH + (M+H)+C2H5+ + RH C2H4 + RH2

+ (M+H)+

2. Alkyl addition:

C2H5+ + RH RH • C2H5

+ (M+29)+

C3H5+ + RH RH • C3H5

+ (M+41)+C3H5 + RH RH C3H5 (M+41)

3. Hydride abstraction : lower PA molecules (M-1)+

CH5+ + RH CH4 + H2 + R+

C2H5+ + RH C2H6 + R+

18

2 5 2 6

• CI Mass spectra of reagent gas ?

19

3.3 Fast Atom Bombardment (FAB) Ion Source( )

Comatrix MatrixCesium ion beam

P

MH+

[ or MX ]+

Proton

X : Na, K, Li[ or MX ] X : Na, K, Li8000Vor 1KV Ion desorbed from matrix

Advantage Soft Ionization(MH+, MNa+...): little fragmentationgEasy adaptation of HRMS: accurate mass measurementMatrix can be useful as ref. ionSample and Matrix

20

1) Schematics of FAB Ion Source

FAB gun

6~10 keV

Primary atom or ion beam

Probe Secondary ions

Through Vacuum Lock

Sample/Matrix To mass analyzerVacuum Lock

Xe Xe+ Xe+ Xe0ionization acceleration neutralization

21

Xe Xe Xe XeSlow atoms Slow ions Fast ions Fast atoms

2) Mechanism of ion formation )

•Impact of a high-energy atom or ionformation of a high-temperature, high density

gas in the cavity that is formed at the point of impact

•Generation of additional ionsd l t di l d it dsecondary electrons, radicals, and exited

neutral species

Mass Spectrom. Rev., 5, 191 (1986)

22

4) Matrix of FAB Ionization

Successful ionization by FAB is deeply dependent on the matrix selection for the analysis.

Matrix requirements

- Dissolve the sample to be analyzed, (usually 1 mg/100 mL)

- Facilitate in the ionization of the sample

- Dissolve the sample to be analyzed, (usually 1 mg/100 mL)

- Facilitate in the ionization of the sample

Matrix requirements

- low volatility

- Not undergo a chemical reaction with the sample.

- Constantly replenish the surface with new sample

- low volatility

- Not undergo a chemical reaction with the sample.

- Constantly replenish the surface with new sampley p p

- Minimize sample damage from the high-energy particle beam

- Reduce damage to the sample by absorbing the impact of the primary beam

- Prolongs the sample ion current by constantly replenishing the upper layer with the fresh sample

y p p

- Minimize sample damage from the high-energy particle beam

- Reduce damage to the sample by absorbing the impact of the primary beam

- Prolongs the sample ion current by constantly replenishing the upper layer with the fresh sample

proton donor(+ve mode)proton donor(+ve mode)Functions

Prolongs the sample ion current by constantly replenishing the upper layer with the fresh sample

- Reduces the binding energy of the sample molecules

Prolongs the sample ion current by constantly replenishing the upper layer with the fresh sample

- Reduces the binding energy of the sample molecules

- proton donor(+ve mode)

- proton acceptor(-ve mode)

- solvent

- sample reservoir

- proton donor(+ve mode)

- proton acceptor(-ve mode)

- solvent

- sample reservoir

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- sample reservoir

- reagent

- sample reservoir

- reagent

Matrix Selection of FAB MS

MatrixMol.

Formula M/Z Characteristics

Glycerol C3H8O3 92 0473 •Best choice for polar compoundsGlycerol C3H8O3 92.0473 •Best choice for polar compounds

1-thioglycerol C3H8O2S 108.0245 •More volatile than glycerol, evaporates quickly

3-nitrobenzyl alcohol (NBA) C7H7NO3 153.0416

•Best choice for less polar compounds and many

organometallics3 nitrobenzyl alcohol (NBA) C7H7NO3 153.0416 organometallics

2-nitrophenyl octyl ether

(NPOE)C14H21NO3 251.1521

•Only FAB matrix with no reactive hydrogen

Triethanolamine C6H15NO3 149.1052•Good matrix for negative-ion FAB

•Enhances [M-H]- formation

magic bullet C4H10S2O2 154.0122

dioctyl phthalate (DOP) C H O 390 54

•Widespread contaminant in solvents, gives characteristic 149

peak in EI mass spectra dioctyl phthalate (DOP) C24H38O4 390.54

•Use care to avoid contaminating the mass spectrometer

[bis(2-ethylhexyl) phthalate] 384

24

sulfuric acid H2SO4 97.967

•Good for some inorganics and organometallics (e.g. copper

phthalocyanine)

•Corrosive, use care

25

• Applications of FAB MSpp

• Synthetic Peptides

• Recombinant DNA Proteins and Glycoproteins

• Carbohydrates

• Drug Metabolites (including intact congugates)

• Detergents (anionic, cationic, non-ionic, amphoteric)Detergents (anionic, cationic, non ionic, amphoteric)

• Biocides

• Petrochemicals• Petrochemicals

• Oil Additives

Oi i C i• Oil Field Chemicals

• Phospholipids

26

3.4 Matrix Assisted Laser Desorption Ionization (MALDI)

Comatrix MatrixLaser

P

MH+

[ or MX ]+

Proton

X : Na, K, Li[ or MX ] X : Na, K, Li

Advantage High Sensitivity Soft Ionization(MH+, MNa+...)( , )Mixture AnalysisLow CostNo Contamination

S l & i

27

Sample & matrix

• Selection of Matrix in MALDI

Sinapinic Acid Proteins >10kDa

a-Cyano-4-Hydroxy- Peptides<10kDaa Cyano 4 Hydroxycinnamic acid

Peptides 10kDa

2,5 Dihydroxybenzoicacid

Neutral Carbohydrates,Synthetic Polymersacid Synthetic Polymers

“Super DHB” Proteins,

Glycosylated proteins

acid

Oligonucleotides

HABA Proteins,

3-Hydroxypicolinic

Oligosaccharides

,

28

• Advantage

– High sensitivity, mass range

– Simple structure

– Easy operation & maintenance, low cost

– Fast acquisition and automation

• Disadvantage

– Accuracy resolution [~10 000]Accuracy, resolution [~10,000]

– delayed extraction, reflectron

– Different calibration for different mass range & experimental g pcondition (laser, matrix)

29

MALDI/TOF

4 I l t d b l t i l fi ld t th

1. Sample is mixed with matrix& dried on target

5. Ions strike thedetector at4. Ions are accelerated by an electrical field to the

same kinetic energy, and they drift (or fly) down afield free flight tube where they are separated inspace.

Flight tube

g

20 - 30 kV

different times,depending on themass to chargeratio of the ion.

High vacuum2. Target is introduced into highvacuum of MS

g

High voltage

•Pulsed laser

3. Sample spot is irradiated with laser,desorbing ions into the gas phase and startingthe clock measuring the time of flight.

Time

6. A data system controls all instrument parameters,acquires the signal vs. time, and permits dataprocessing

30

processing.

Reflector for TOFReflector

Higher resolutionHigher mass accuracyPSD(Post Source Decay) First Detector

Reflector(Ion mirror)

PSD(Post Source Decay)Second Detector

Ion Gate

Laser

The reflector increases the overall path length for an ion and it corrects for minor variation in the energy spread of ions of the same mass Both effects improve

31

variation in the energy spread of ions of the same mass. Both effects improve resolution.

• Applications of MALDI/TOF-MSApplications of MALDI/TOF MS

Molecular biology, biochemistry

– Biopolymer CharacterizationBiopolymer Characterization

• Protein & peptides [proteomics]– Sequence Analysis

• DNA, RNA [genomics]– In- Source Decay

– Exonuclease Sequencing– Exonuclease Sequencing

• Carbohydrate

• Lipid

Pharmaceutical Industry

– Combinatorial Chemistry

Ch i l I dChemical Industry

– Synthetic Polymer Characterization

32

3.5 Atmospheric Pressure Chemical Ionization (APCI)

S l I l t N lSample Inlet Nozzle(Lower Voltage)Pressure = 760 torr

MH+ MH+ MH+S l t Fl F HPLC

N2

MH+

MH+ MH MHSolvent Flow From HPLC

MH+

Heat applied to metal sheath

Corona discharge needle

Heat

to metal sheath needle

33

APCI

Simple and efficient device for the LC/MS analysis of compounds

Needle : 3 6kV potential creates a corona dischargeNeedle : 3-6kV potential creates a corona discharge

Ionization : by CISoft ionization which generates pseudo molecular ions

MH+ (M NH )+• MH+, (M+NH4)+ etc.

• (M-H)-, (M+CH3COO)-, (M+Cl)- etc

Generates singly charged ions, in general

34

Suitable for less polar compounds compared to ESI

Same degradation may occur in case of labile compounds

• Characteristics of APCI

• Both positive and negative ions can preferentially be formed in the

APCI source using a corona dischargeAPCI source using a corona discharge

• Ion – molecule reaction

• Positive ion mode

- the creation of reactant positive ions in ambient air

(the proton hydrates, H3O+[H2O]n)

- the major primary ions N2+, O2

+, H2O+ and NO+ are formed by electron

impact of corona-created electrons on the major neutral components in air

- Proton transfer reaction H3O+[H2O]n + T TH+ (H2O)m + (n-m+1)H2O

if the gas basicity (proton affinity) of T is greater than that of water

TH+ (H2O)m TH+ by stripped off in the gas curtain and declustering lens region

35

lens region

I i i d• In negative ion mode,- electron created by the corona are rapidly thermalized (lose excess energy) th h lli i ith t l d t d b l t ti i h Othrough collision with neutrals and captured by electronegative species such as O2

to form O2- and O-.

- super oxide (O2-) and its hydrates (O2

-[H2O]n) and cluster (O2- [O2]n)p ( 2 ) y ( 2 2 n) ( 2 2 n)

Ion – Molecule Reaction

• Charge transfer

R+ + T T+ + R

R- + T T- + R

• Proton transfer

RH+ + T TH+ + R

R- + TH T- + RH

36

APCI – MS spectra of Phenylbutazonesp y

37

APCI –MS/MS spectra

38

3.6 Electrospray Ionization (ESI)3.6 Electrospray Ionization (ESI)

39

1) Characteristics of ESISoft ionization, which generates pseudo molecular ions

• MH+, (M+NH4)+, (M+Na)+, (M+K)+ etc.MH , (M+NH4) , (M+Na) , (M+K) etc.

• (M-H)-, (M+CH3COO)-, (M+Cl)- etc.

Applicable for wide range of compounds with relatively high sensitivity

• Up to 150 kDa in case of proteins

• Middle to High polar compounds

• Thermally labile compounds

• Non covalently binding complex

Generates multiply charged ions of biopolymers

• (M+nH)n+ / (M-nH)n-

40

•ESI Mass Spectra

1picomole Myoglobin

MW: 16 939 4 amMW: 16,939.4 amu

LC/MS analysis m/z 1230

Heme dimer

Second series protein + Heme

Synthetic DNA

MW 4260 7MW: 4260.7 amu

Negative ion mode

41

Ch 14. Basics of Mass Spectrometry II: m/z analysis & MS spectra

• All analyzers sort ions based on their mass-to-charge ratios (m/z)

Magnetic Sector

TOFTOF

1970s/80s

1990s

Q-TOF

1950s/60s1980s/90s

1990s-2000s-

Ion TrapsQuadrupoles

42

1. Analyzer

• Ability to separate ionsy p

• High accuracy, High sensitivity

• High mass range

43

g g

• Structural information

1.1 Time of Flight (TOF)g

• TOF commonly used with MALDI

• Different velocity

• Reflectron improves Resolution• Reduce KE distribution of ions

y

• KE = 1/2mv2 v = (2KE/m)1/2

44

• Reduce KE distribution of ions• Expense of sensitivity• Limited mass range

Assumed with Ions moving with the same initial E /Assumed with Ions moving with the same initial E zm /ions produced by pulse

Potential energy = z V

= neV

n: number of chargese: charge of electron

neV

Kinetic energy = ½ mv2m: ion massv: velocity

1

2

2

12

1mvzV

22/

Vt2)/(

2

1 tLm 2

/L

zm

2/ tzm / tzm TOF with MALDI: widespread applications in protein, DNA fragment

45

monoisotopic peptide MW<20 ppm (+ 0.2 Da for 1000Da)

1.2 Magnetic Sector

46

Ions formed continuously are accelerated toward detectoryby electrical potential V

Under magnetic forcecentripetal force : Bzv B: magnetic field

r

mvBzv

2

centrifugal force :

z: charge of ionv: ion velocity

r

mv 2 Brzm

r

/r v

kinetic E of moving ion at magnet =2mvg g

potential energy = zV

2

m

zVv

2

rBzm /

22

under const. V, scan B

only specific m/z passes

47

Vzm

2/ – only specific m/z passes

1.3 Quadrupole

most widely used with GC – organic compd.

opposing poles are connected to RF & dc generators

RF/DC = Constant

48

-ouput of RF field,energy delivered as sinusoidal wave /c

: RF frequencyf d V V U : amplitude-ouput of dc generator, +V, -V

Resulting two variables

22

8

mr

zUa U: amplitude

V: applied dc voltage

22

4

mr

zVq

mr

V: applied dc voltager: radius of circle tangent to the inner

surface of the quadrupoemr

a, q have no physical meaning, solution of d.eqn.

If r are constant and a/q= by instrumentU2If r, are constant, and a/q= by instrumentV

8/

Uzm or

4V

49

22/

arzm or 22qr

4/

Vzm

22/

qrzm

m/z : varies linearly with Veasy to control instrumentally

to vary rapidly over a scan with little or no lag timebetween scans

Mass Selective Detector (MSD) in GC/MSMass Selective Detector (MSD) in GC/MS

dc generator: 0~200 Vdc generator: 0 200 VRF generator: 0~1200 Vupper limit : 800~1000 in GC/MSpp

~2000 in LC/MS

50

Selective Ion Monitoring (SIM)Selective Ion Monitoring (SIM)

: programmed to produce only discrete U or V.

- only specific m/z traverse the analyzer & all others rejected

advantage: enhanced selectivity for detecting low conc. compounds

User selects only few ions (~3): abundant and characteristic structure by increasing y gdwell time (at specific U or V) fraction increases (nanogram to picogram level)

51

1.2 Ion Trap (quadrupole ion trap)

RFz0

ac or dc supplied

0

r0

supplied

- Applicable to ESI- Tandem mass spectrometry

52

p y

ac or dc supplied

RFz0

r0ac or dc supplied

RFz0

r0

suppliedsupplied

- Ions are trapped in concentric, three dimensional orbits (according to m/z) around the center of the ion trap(according to m/z) around the center of the ion trap.

8zU22

0

4

8

zV

mr

zUaz V: dc voltage to end caps

U: amplitude of RF voltage to ring

220

4

mr

zVqz z0: distance

53

- For any given value of q Uzm4

/ For any given value of qz Uqr

zmz

220

/

when instrument scans from low to high Uwhen instrument scans from low to high U, ion motion with higher m/z oscillates larger and largerin z-direction until they finally escape the trapped ion cloudy y p ppand exit to detector.

-more sensitive than transmission quadrupole -mass range : up to m/z 70,000

54

General Comparison of Mass Analyzers

Mass range limited to about 3000 m/zi i A

Ease of switching between positive/negative ionS i

Range m/z 3000i 2000

Quadrupole

DisadvantagesAdvantagesmass range

and resolutionMass analyzer

Poor adaptability to MALDISmall sizeRelatively low costSmall sizeMedium resolution

Resolution 2000Q p

Si l d i l tR / 2000 Li it d f t i lI Simple design, low costWell-suited for tandem mass spectrometry(MSn , n≤4)Easy for positive/negative ions

Range m/z 2000Resolution 1500

Not tolerant of high pressureCapable of high resolutionRange m/z 20,000Magnetic sector

Limited mass range of current commercial versions;however, progress is being made in their development

Ion trap

Low resolutionDiffi lt f d t ti t l t

Highest mass rangeV f t d

Range m/z ∞R l ti 350

Time-of-flight(TOF)

g pExpensiveInstrumentation is massiveRelatively slow scanning

p gCapable of exact massMedium mass rangeCan be very reliable,manufacturer dependent

g ,Resolution 10,000

Magnetic sector

Good resolving power has limited m/z rangeLower sensitivity than TOF

Good resolutionVery fast scan speed

Range m/z ∞Resolution 1500

Time-of-flight f t

Difficulty of adaptation to electrospray Very fast scan speedSimple design,low costEase of adaptation to MALDI

Resolution 350

High vacuum (<10-7 Torr) requiredSuperconducting magnet required, expensive

High resolutionWell-suited for tandom mass spectrometry(MSn , n≤4)

Range m/z 10,000Resolution 30,000

Fourier transform-mass spectrometry

yy pSimple design,low costrefrectron

55

pInstrumentation massive

( , ≤ )(FT-MS)

2. Other Analysis: Tandem Mass Spectrometry(MS/MS)(MS/MS)

Fragmentation induced by ion-molecule collision (CID)

56

Tandem Mass SpectrometryTandem Mass Spectrometry

MS 1 MS 2

Collision CellP1

P2

Tandem MS

MS-1 MS-2Hegas

2

P3

P4

P5

ES Source D t t

Select for a particular ion

HPLC

F1 F2 F3 F4 F5

ES Source Detector

Input: peptides from enzymatic digest

particular ion(peptide)

Output: fragments fromdaughter ionsy g daughter ions

57

CID (Collision Induced Dissociation) Patternof a Tryptic Peptide

b series b1

114.1b2

261.2b3

348.2b4

476.3b5

575.3b6

632.3[LFSQVGK+H]+

of a Tryptic Peptide

L F S Q V G Kions

665.4y

518.3 431.3 303.2 204.1 147.1

[LFSQVGK+H]+

=778.4 Da

y series ionsy6 y5 y4 y3 y2 y1

b

K G V Q S FCID

b2

y6

y5spectrum

b4b5

y6

y

b3b6

y4y3y2

y1

58m/z

59

60

61

62

3. Detectors

63

3.1 Faraday Cup

• Create a current

• Induce several 2nd electron

• Small amplification of signal

• Relatively insensitive

• Simple in design

64

• Simple in design

3.2 Electron Multiplier

65

3.3 Photomultiplier

-Ion initially strike a dynode resulting in the emission of e- strike-Ion initially strike a dynode, resulting in the emission of e strike

a phosphorus screen, release photons

-PM tube is sealed in vacuum

66

-Lifetime is 5-year or greater

High energy electron multiplier

• Ions accelerate into EM, producing more secondary ions o s acce e ate to , p oduc g o e seco da y o s

• HED serve to increase ion E and therefore the signal intensity resulting in greater sensitivity

67

68