tandem ms = ms / ms - boku · tandem ms = ms / ms in tandem ms (msms ) (pseudo -)molecular ions are...

Tandem MS = MS / MS

In tandem MS (MSMS)

(pseudo-)molecular ions are selected in MS1

ESI-MS give information on the mass of a molecule

but none on the structure

(pseudo-)molecular ions are selected in MS1

and fragmented by collision with gas.collision induced decay – CID

electron transfer decay – ETD (= ECD)

The fragment ions are analyzed in a second MS.

Quadrupole

ESIion

source

Entranceoptics

Massanalyzer

detector

Q0

Q1

Separation of

primary ionsMS

Triple Quadrupole

Collision cell

ESIion

source

Q2

Entranceoptics detector

Q0

Q1 Q3

Separation of

primary ionsMS ion transfer

Separation of

fragment ionsMS / MS

tandem MS

Precursor

ion selection

Difference: Collision energy

just a longer flight path, nothing gained

Target analysis by MS-MS

Collision cell

ESIion

source

Q2


Q0

Q1 Q3HPLC

Analysis of

fragment ions

MS / MS

Precursor

ion selection

In LC-MS: mass selected

for compounds eluting in a

certain time window

Target analysis by MS-MS

Collision cell

ESIion

source

Q2


Q0

Q1 Q3HPLC

Detection of

specific

fragment ionsMS / MS

Precursor

ion selection

-

fixed in

time window

Only m/z 147Example: dexamethazon m/z 393

Or: 121,147,237“Transitions“

Reaction monitoring

SRM / MRM

Tandem MS = MS / MS

Target analysis: mass of analyte and mass of fragments are

known beforehand.

MS1 and MS2 are preset on target masses

maximum dwell time, maximum sensitivity

In proteomics applications nothing is known. Precursor mass is

determined by “survey scan“

Precursor mass is selected by operator (off-line) or PC (on-line;

“data-dependent acquisiton“) according to abundance, charge

state and additional information

Tandem MS = MS / MS

Precursor Ion scan:

Neutral loss scan:

Fragment masses indicate structural details

e.g. 365 reveals glycopeptides

Loss of a certain mass by removal of chemical group, e.g. – 18 by H2O

Loss of 98 indicates phosphorylation

Requirement for proteomics applications:

Resolution of multiply charged isotope clusters, high accuracy of MSMS

→ Q-TOF, ion trap, IT-ICR

MS1

quadrupole

MS2

TOFentrancelenses

Collision celloctapole

Hybrid-instruments: Quadrupole-TOF (Q-TOF)

rotary vacuum pumps „rough pumps“

Waters Synapt II:

R in V-mode: 20.000

R in W-mode: 40.000

TOF as MS2 allows

higher resolution, accuracy and

upper mass limit.

rotary vacuum pumps „rough pumps“

turbomolecular pumps for high vacuum inside instrument

PC for control and data aquisition

Server for databank searches

N2-generator (and oil-free compressor)

Argon (collision gas)

Bruker Maxis 4G:

up to 60.000

Hybrid-instruments with Orbitrap analyzers

Combination of ion trap

and Orbitrap analyzer

Newest option:

Combination of quadrupole

with Orbitrap analyzer

Exact mass analysis of unknown compounds

over a wide mass.

Typical application:

Applications of MS-MS

Hybrid instruments or Trap:

Typical application:

peptide identification by MS-MS

structural analysis of biochemicals ....

---> fast "scan" rate of TOF or Trap

MS1 MS2

Q-TOF in MS Mode

entrancelenses

octapole

Primary ions are

collected and sent to

MS1

MS1 MS2

Q-TOF in MS Mode

MS1 does not filter,

all ions pass through

MS1 MS2

Q-TOF in MS Mode

collision cell is inactiv

(ions are slow)

ions pass unaltered

MS1 MS2

Q-TOF in MS Mode

TOF analyses

primary ions

MS1 MS2

Q-TOF in MS/MS Mode

entrancelenses

octapole

Primary ions are

collected and sent to

MS1

MS1 MS2entrancelenses

Collision cell

Q-TOF in MS/MS Mode

MS1 selects parent

ion of a certain mass

(m/z);Others cannot pass


Collision cell

Q-TOF in MS/MS Mode

Collision with gas atoms

(e.g. Ar) causes

fragmentation of ions

(collission induced

dissocation = CID)

Collision energy is controlled

by kinetic energy of the

analyte ions.


Collision cell

Q-TOF in MS/MS Mode

Daughter ions leave

the collision cell


Collision cell

Q-TOF in MS/MS Mode

MS2 (TOF) analyses

fragment ions

De novo sequencing of a peptide of mass 1212.33 from a wasp venom allergen

Proteomics work with ESI-MS/MS

MSMS 607.33 ES+

%

v ulgaris PLA MSMSv ulgaris_PLA _MSMS Max Ent 3 68 [Ev 4631,It50,En1] (0.040,200.00,0.060,14 00.00,2,Cmp)

AV I Y M A E C IK

KI C E A M Y I V A

y6y8

yMax

bMax

767.361043.51

866.45

+

AVIYMAECLK

VIYMAECLK

IYMAECLK+

++

1 00 200 300 400 50 0 600 700 800 900 10 00 1100 1200 1300

M/z0

y5

y7

y8

theoret.

[MH]+

[MH ]2

2+

420.26 549.29

620.33

930.43

y3b3

249.17

239.18172.12120.07

284.21

361.13311 .11

y4

522.22465.67 601.38

703.37

621.26704.32

768.42

867.38

929.36

979.51

1044.59

1239.771149.671045.44

1283.761299 .69

+

IYMAECLK

YMAECLK

MAECLK

AECLK

+

+

+ECLK

+CLK

+LK

Doubly charged precursor → singly charged fragments

Proteomics work with ESI-MS/MS

100

2: TOF MSMS 1087.35ES+ 1553.54

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200mass0

100

%

MSMS 1087.35 ES+ 1553.54

1438.431236.321050.31

308.08

280.10201.11

1034.38890.34819.24

490.17330.15 599.23508.16

721.27762.24 963.33 1147.51

1337.421578.42

1476.68

1249.23 1753.60

1666.521866.71

2179.862067.54

113

115

77

200 M 2173.70exp


100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200mass0

100

%

1553.54

1438.431236.321050.31

308.08

280.10201.11

1034.38890.34819.24

490.17330.15 599.23508.16

721.27762.24 963.33

1147.51

1146.40 1235.47

1337.421578.42

1249.68

1276.68

1249.23 1753.60

1666.52

1578.771866.71

2179.862067.542185.86

FC+H+

W T TD I S

IAG

C

Y

FCISIDTTWCAGYCYTR

Peptide fragmentation

Major fragments derived from a peptide (protonated)

C C C CH N N N N

R1 R2 R3 R4

C C C COOH

y2y1y3

+ 2 H + 2 H+ 2 H

C C C C2

H N N N NC C C COOH

O O OH H H HH H H

b2b3b1 a2

a3a1


Fragmentation of a singly charged peptide

C C C CC C C

R

H2N N N NN N N

R1 R2 R3 R5 R6R4

NH3

C C CC C C COOH

O O OO O OH H H HH H HH H HH H H

y2 y1y3y4y5y6

C C C CC C C

R

H N N N NN N N

R1 R2 R3 R5 R6R4

NH2

C C CC C C COOHC C C CC C CH2N N N NN N NC C CC C C COOH

O O OO O OH H H HH H HH H HH H H H

C C

CH2N N N

R1 R2

R3

C C C

O O

O

H HH H

CC C

R

CNN N N

R5 R6R4

NH2

C C C COOH

O O OHH H HHH

H

H

H H

y4-ionneutral b-fragment

Fragmentation of a doubly charged peptide

C C C CC C C

R

H3N N N NN N N

R1 R2 R3 R5 R6R4

NH3

C C CC C C COOH

O O OO O OH H H HH H HH H HH H H

C C C CC C C

R

H N N N NN N N

R1 R2 R3 R5 R6R4

NH2

C C CC C C COOHC C C CC C CH3N N N NN N NC C CC C C COOH

O O OO O OH H H HH H HH H HH H H H

CC C

R

CNN N N

R5 R6R4

NH2

C C C COOH

O O OHH H HHH

H

H

H H

y3-ion

peptide mass + H

b3-ion

C C

CH3N N N

R1 R2

R3

C C C

O O

O

H HH H


acylium ion

Peptide fragmentation

b3-ions

peptide mass - 17

C C

CH3N N N

R1 R2

R3

C C C

O O

O

H HH H

C C CH2N N N

R1 R2 R3

C C C

O O

O

H H HH H

C C CH2N N N

R1 R2 R3

C C

O OH H HH H

a3-ion

peptide mass - 45

CH2N

R3

H

immonium ion

reveal amino acids in peptide

Triple quads, ion traps, Q-Tofs and similar mass specs can only provide

low energy (eV – range) fragmentations.

can be modulated within certain range (adjusted to mass of peptide)

Collision energy

Collision induced or collison activated dissociation of parent ions

(CID or CAD)

Yields relatively simple fragment spectra.

Disadvantage: Leu and Ile cannot be discriminated

High energy CID in BE or TOF-TOF instruments.

PBC m/z 300 – 2200, all MS

BSA 100fmol, Time=43.0-43.3 min (#339#341), 100%=159692arb

MS

y2

b3

y3y4

y5

b7

y7

b8

y6

b9

y9

y10

b11

b12

MS/MS

Applications of nano-LC / MS-MS

all MS/MS

100 fmol BSA injected on column

30 40 50 Time [min]

m/z600200 1000

Result of the

database search of

silver stained

protein gel spot.

Archeal histon was

unambiguously

found in a Archeal histon

Protein identification by LC / MS-MS

found in a

Halobacterium

salinarum (genome)

database using

MASCOTTM.

Important software packages for protein identification:

MASCOT, GPM, SEQUEST …. and company derivatives e.g. MassLynx, Proteome Discoverer

ProteinScape …..

Data-dependant aquisition (DDA)

At first, the machine works in the MS mode (survey mode) until

mass is detected that is:

- of sufficient intensity

- not in exclude list (background, trypsin, keratin)

- doubly or triply charged

(- is in include list )

Then, machine switches into MS/MS mode to acquire CID spectrum of

this compound for e.g. ca. 1 sec

Then, this mass is “locked” for some time to prevent redundancy.

Often, the survey mode detects more than one signal � MSMS 1, MSMS 2, etc.

before switched back to survey.

MS/MS specials

I.) Dependancy of ion type and collision energy

- the larger the more energy required

- charges help fragmentation

- careful choice of collision energy profile

II.) DDA tends to overlook many peptidesII.) DDA tends to overlook many peptides

Solutions: - increase speed of instrument

- optimize selection criteria

- rerun sample with inclusion list and/or exclusion list

Quality criterion: % of identifiable peptide spectra

Notorious problem: hybrid spectra (precursor selection not rigorous)

MS/MS specials

Precursor selection problem turned upside down: MS/MSALL

(in segments: SWATH)

MS/MS specials

The score depends on:

1.) number of peptides fitting to a particular peptide

“one hit wonders“ usually disregarded

2.) number of fragments fitting to theoretical digest of peptide

Searching for peptide fragment (and mass) in data banks e.g. „Swissprot“

By MASCOT or SEQUEST and related tools yields list of hits with probability

2.) number of fragments fitting to theoretical digest of peptide

(also: those NOT fitting)

3.) size of peptide (the larger, the better)

4.) size of protein (the smaller the better)

5.) allowance of missed cleavage sites

6.) allowance of modifications � the more the worse (search space !)

7.) size of databank (too small is bad !)

Sample inlet systems for ESI

Syringe pump

5 to 50 µL / min

System testCalibration

“Simple” samples

Mosttypical LC-MSapplications

(pharmaceutical,environmental,

forensicetc.)

Liquid chromatography

50 to 1000 µL / min


For limited sampleamounts in bioscience

1-2 µL of samplegive 30 min

of analysis time

Nanospray tips

20 nL /min

40 mm

For demanding life scienceapplications

Nanoflow LC

100 to 1000 nL / min

split

Column i.d. Flow rate Technique

4.0 mm 1.0 mL/min Conventional HPLC

2.0 mm 0.25 mL/min Small bore LC


1.0 mm 0,0625 mL/min Micro LC

75 µm 350 nL/min Nano LC

1.0 mm 0,0625 mL/min Micro LC

300 µm 5.6 µL/min Capillary LC

180 µm 2.0 µL/min Capillary LC

S/N = 1

1.0 ml/min

4.6 mm i.d.

S/N= 3800


1.0 ml/min

75 µm i.d.

Signal to

Noise ratio


4.6 mm i.d

1.0 mm i.d.

300 µm i.d.

UV 206 nm

ESI has

2 pmol digested myoglobin (each column)

300 µm i.d.

75 µm i.d.

ESI has

a similar, even

stronger

concentration

dependance

Chromatographiesäulen im Vergleich

4 mm

0.18 mm

75 µm

Nano-Elektrospray

Nano-Elektrospray

Diameter 1 mm

Diameter 4 mm, Flow rate 1.6 mL / min

Diameter 0.32 mm

Diameter 0.075 mm

Flow rates ?

tandem ms = ms / ms - boku · tandem ms = ms / ms in tandem ms (msms ) (pseudo -)molecular ions are...

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