Tandem MS = MS / MS

ESI-MS give information on the mass of a molecule but none on the structure

In tandem MS (MSMS) (pseudo-)molecular ions are selected in MS1 and fragmented by collision with gas.
collision induced decay CID = collision activated decay CAD (plus other methods)

The fragment ions are analyzed in a second MS.

Quadrupole
Entrance optics Mass analyzer

ESI ion source

Q0

Q1

detector

MS

Separation of primary ions

Triple Quadrupole
ESI ion source
Entrance optics

Q0

Q1

Collision cell Q2

Q3

detector

MS MS / MS
tandem MS

ion transfer
just a longer flight path, nothing gained

Separation of primary ions

Precursor ion selection

Separation of fragment ions

Difference: Collision energy

Target analysis by MS-MS

ESI HPLC ion source
Entrance optics

Q0

Q1

Collision cell Q2

Q3

detector

Precursor ion selection

Analysis of fragment ions

MS / MS
In LC-MS: mass selected for compounds eluting in a certain time window

Target analysis by MS-MS

ESI HPLC ion source
Entrance optics

Q0

Q1

Collision cell Q2

Q3

detector

MS / MS

Precursor ion selection fixed in time window

m/z 393

Detection of specific fragment ions

Example: dexamethazon

Only m/z 147

Transitions

Or: 121,147,237

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:
Fragment masses indicate structural details e.g. 365 reveals glycopeptides

Neutral loss scan:

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

Q-TOF
Description of system

2 rotary vacuum pumps 3 turbomolecular pumps Embedded PC PC for control and data aquisition Server for databank searches N2-tenerator Argon (collisiongas)

Quadrupole-TOF (Q-TOF)
entrance lenses

MS1 quadrupole

Collision cell
octapole

MS2 TOF

TOF as MS2 allows higher resolution, accuracy and upper mass limit.

R in V-mode: 10.000 R in W-mode: 17.500

Applications of MS-MS
Hybrid instruments or Trap: Exact mass analysis of unknown compounds over a wide mass. Typical application: peptide identification by MS-MS structural analysis of biochemicals .... ---> fast "scan" rate of TOF or Trap

Q-TOF in MS Mode
entrance lenses

MS1

octapole

MS2

Primary ions are collected and sent to MS1

Q-TOF in MS Mode
MS1 MS2

MS1 does not filter, all ions pass through

Q-TOF in MS Mode
MS1 MS2

collision cell is inactiv

(ions are slow)

ions pass unaltered

Q-TOF in MS Mode
MS1 MS2

TOF analyses primary ions

Q-TOF in MS/MS Mode

entrance lenses

MS1

octapole

MS2

Primary ions are collected and sent to MS1

Q-TOF in MS/MS Mode

entrance lenses

MS1

Collision cell

MS2

MS1 selects parent ion of a certain mass (m/z);

Others cannot pass

Q-TOF in MS/MS Mode

entrance lenses

MS1

Collision cell

MS2

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.

Q-TOF in MS/MS Mode

entrance lenses

MS1

Collision cell

MS2

Daughter ions leave the collision cell

Q-TOF in MS/MS Mode

entrance lenses

MS1

Collision cell

MS2

MS2 (TOF) analyses fragment ions

Proteomics work with ESI-MS/MS

De novo sequencing of a peptide of mass 1212.33 from a wasp venom allergen
v ulgaris P MSMS LA v ulgaris _PLA _MSMS Max Ent 3 68 [E 4631,It50,En1] (0.040,200.00,0.060,14 00.00,2,Cmp) v

MSMS 607.33 ES+

E C IK

AV I

bMax V A yMax

KI

120.07 172.12
0 1 00

+AVIYMAECLK+ + VIYMAECLK 767.36 y6 1043.51 + 866.45 y8 IYMAECLK 620.33 + y5 YMAECLK + MAECLK theoret. + [MH] 979.51 AECLK 930.43 703.37 y7 + ECLK 420.26 549.29 1044.59 284.21 y4 y3 + 249.17 867.38 2+ b3 [MH 2] CLK 768.42 1239.77 704.32 1045.44 1149.67 361.13 601.38 465.67 522.22 + 239.18 621.26 311 .11 929.36 1283.761299 .69 LK M/z
+

200

300

400

50 0

600

700

800

900

10 00

1100

1200

1300

Doubly charged precursor singly charged fragments

Peptide fragmentation
Major fragments derived from a peptide (protonated)

y3
+2H

y2
+2H

y1
+2H

R1 H2N C C N
H

R2 C C N
H

R3 C C N
H

R4 C COOH
H

a 1 b1

a 2 b2

a 3 b3

Doubly charged precursor singly charged fragments

Fragmentation of a singly charged peptide

R1 H2N

y6

R2

y5

R3

y4

R4

y3

R5

y2

R6 C
H

y1
C N O
H

NH3 R C COOH
H

C C N
H

C C N
H

C C N
H

C C N
H

C C N
H

NH2 R1 H2N C C N
H

R2 C C N
H

R3 C C N
H

R4 C C N
H

R5 C C N
H

R6 C
H

R C N O
H

C COOH
H

HH

NH2
R1 H2N C C N
H

R2 C C N
H

R4 C C N
H

R5 C C N
H

R6 C
H

O C C R3
H

R C N O
H

N
H

C COOH
H

neutral b-fragment

y4-ion

Fragmentation of a doubly charged peptide

NH3 R1 H3N C C N
H

R2 C C N
H

R3 C C N
H

R4 C C N
H

R5 C C N
H

R6 C
H

R C N O
H

C COOH
H

NH2 R1 H3N C C N
H

R2 C C N
H

R3 C C N
H

R4 C C N
H

R5 C C N
H

R6 C
H

R C N O
H

C COOH
H

HH

Doubly charged precursor singly charged fragments

R1 H3N C C N
H

R2 C C N
H

NH2
O C C R3
H H

R4 C C N
H

R5 C C N
H

R6 C
H

R C N O
H

N
H

C COOH
H

b3-ion

y3-ion
peptide mass + H

Peptide fragmentation
R1 H3N C C N
H

R2 C C N
H

O C C R3

R1 H2N C C N
H

R2 C C N
H

R3 C C O
H

b3-ions
peptide mass - 17
R3 H2N C
H

acylium ion

R1 H2N C C N
H

R2 C C N
H

R3 C
H

a3-ion
peptide mass - 45

immonium ion
reveal amino acids in peptide

Collision energy
Collision induced or collison activated dissociation of parent ions (CID or CAD)
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) Yields relatively simple fragment spectra.

Disadvantage: Leu and Ile cannot be discriminated High energy CID in BE or TOF-TOF instruments.

Applications of nano-LC / MS-MS

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

MS MS/MS
y5 y6 b8 b9 y9

PBC m/z 300 2200, all MS

b3 y2 200

y3

b11 y10 b12

y4 b7

y7

600

1000

m/z

all MS/MS

30

40

50

Time [min]

100 fmol BSA injected on column

Protein identification by LC / MS-MS

Archeal histon

Result of the database search of silver stained protein gel spot. Archeal histon was unambiguously found in a Halobacterium salinarum (genome) database using MASCOTTM.

Data-dependant aquisition
At first, the machine works in the MS mode (survey mode) until mass is detected which: - is of sufficient intensity - is not in exclude list (not background, not trypsin, not keratin) - is doubly or triply charged - or is in include list Then, machine switches into MS/MS mode to acquire CID spectrum of this compound for e.g. 10 sec Then, this mass is locked for some time to prevent redundancy. Often, the survey mode detects more than one signal before switched back to survey. MSMS 1, MSMS 2, etc.

Sample inlet systems for ESI

Syringe pump 5 to 50 L / min System test Calibration Simple samples

Liquid chromatography 50 to 1000 L / min

Most typical LC-MS applications (pharmaceutical, environmental, forensic etc.)

Sample inlet systems for ESI

Nanospray tips 20 nL /min 1-2 L of sample give 30 min of analysis time 40 mm For limited sample amounts in bioscience

Nanoflow LC 100 to 1000 nL / min

For demanding life science applications

split

Sample inlet systems for ESI

Column i.d. 4.0 mm 2.0 mm 1.0 mm 300 m 180 m Flow rate 1.0 mL/min 0.25 mL/min 0,0625 mL/min 5.6 L/min 2.0 L/min Technique Conventional HPLC Small bore LC Micro LC Capillary LC Capillary LC

75 m

350 nL/min

Nano LC

Sample inlet systems for ESI

S/N= 3800

S/N = 1

4.6 mm i.d.
1.0 ml/min

75 m i.d.

Sample inlet systems for ESI

4.6 mm i.d

1.0 mm i.d.

UV 206 nm
300 m i.d. ESI has a similar, even stronger concentration dependance

75 m i.d.

2 pmol digested myoglobin (each column)

Chromatographiesulen im Vergleich

4 mm

0.18 mm 75 m

Nano-Elektrospray