What does a mass spectrometer do? 1. It measures mass better than any other technique.

2. It can give information about chemical structures.

What are mass measurements good for? To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers

Experimental investigations on the conduction of electricity by gases led to the discovery of the electron in 1897, for which Thomson was awarded the and to the1906 Nobel Prize in Physics. Thomson J.J. On the Masses of the Ions in Gases at Low Pressures Philosophical Magazine, 1899, 48:295, p.547-567. Thomson observed that the new technique could be a used profitably by chemists to analyze chemicals. Thomson J.J. On Rays of Positive Electricity. The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science, 1907, XLVII.

The technique of mass spectrometry had its beginnings in J.J. Thomson's vacuum tube.

F = qE = ma y = ½ at2 L = vt

0

y

x -

Consider a Charged Particle in an Electric Field

_ _ _ _ _ _ _ _ _ _ _ _ _

+ + + + + + + + + + + + +

Magnetic Mass Analyzer

m z

r2 B2

2V =

Fb = qv X B

0

y

x

x x x x

x x x x

m z

r2 B2

2E =

v = E/B

Crossed Fields can be Used as a Velocity Selector

How does a mass spectrometer work?

Ion source: makes ions

Mass analyzer: separates ions

Mass spectrum: presents

information

Sample

Inlet Ion source

Mass Analyzer Detector

Data System

High Vacuum System

Mass Spectrometer Block Diagram

Inlet Ion Source

Mass Analyzer Detector

Data System

High Vacuum System

HPLC Flow injection Sample plate

Sample Introduction

Inlet Ion Source

Mass Analyzer

Detector Data System

High Vacuum System

MALDI ESI FAB LSIMS EI CI

Ion Source

Protonation of Peptides

High voltage applied to metal sheath (~4 kV)

Sample Inlet Nozzle (Lower Voltage)

Charged droplets

+ + + + +

+

+ + + + +

+

+ + + + +

+ + + +

+ + + + + +

+ + + + +

+ +

+ +

+

+ + + + + +

+ + +

MH+

MH3+

MH2+

Pressure = 1 atm Inner tube diam. = 100 um

Sample in solution

N2

N2 gas

Partial vacuum

Electrospray ionization:

Ion Sources make ions from sample molecules (Ions are easier to detect than neutral molecules.)

Electrospray Ionization (ESI)

Vacuum Atmospheric pressure

Mass Analyzer

N2 curtain gas

+HV

nebulizing gas

sample solution

+ + +

+ +

+ + + +

+ + +

+ +

+ +

[M + nH]n+ + +

1. Solvent evaporation 2. Coulombic repulsion

Electrospray Ionization (ESI) Process(Positive Mode)

Ions

1-5 kVpower supply

+

Spraycurrent ( ) i

+

Taylor coneNeedle

Site ofoxidation Site of

reduction

+

++

+ +

+

+

++

++ + + +

++++++

+

++

++ + ++

++

++ ++

+ +++

++++

++++ +

++++ +

++++ +

+++

+++

+++

+++

++

+

++

++

+

++

+

Massspectrometer

+

++

+

++

+ +

+

+

+

+

+

Solvent andneutralized ions

++++ +

++++ +

++++ +

+++

+++

+++

+++

++++ +

+++

+++

+++

+++

+++

+++

+++

+

++

+

+

+++++ +

+++

++

++ ++

+ ++ +

ESI droplets

Adapted from Kebarle, P and Tang, L, , , , 972AAnal. Chem. 65(22)1993

5 µm

Electrospray Ionization (ESI)

hν Laser

1. Sample is mixed with matrix (X) and dried on plate.

2. Laser flash ionizes matrix molecules.

3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X.

MH+

MALDI: Matrix Assisted Laser Desorption Ionization

+/- 20 kV Grid (0 V)

Sample plate

MALDI generation of ions (Matrix-assisted laser desorption ionization)

Sample mixed with a UV-absorbing matrix and is allowed to co-crystallize on the metal target.

Laser pulse (337 nm)

Peptide/protein deposited on crystal surface

[M+H]+

[M-H]-

matrix ions

Matrices for MALDI analysis

Peptides/proteins - 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) - α-cyano-4-hydroxycinnamic acid (CHCA) - 2,5-dihydroxybenzoic acid (DHB) - 2-(4-hydroxyphenylazo)-benzoic acid (HABA) Oligonucleotides - 2-aminobenzoic acid - 3-hydroxypicolinic acid (3-HPA) - 2,4,6-trihydroxyacetophenone (THAP) The choice of matrix depends greatly on the solute to be analyzed.

Inlet Ion source

Mass Analyzer Detector

Data System

High Vacuum System

Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FT-ICR MS Orbitrap MS

Mass Analyzer

•Operate under high vacuum (keeps ions from bumping into gas molecules)

•Actually measure mass-to-charge ratio of ions (m/z) The importance of the mass-to-charge ratio is that according to classical electrodynamics two particles with the same mass-to-charge ratio move in the same path in a vacuum when subjected to the same electric and magnetic fields.

•F=ma (Newton’s second law of motion)

•F=q(E + v × B) (Lorentz force Law)

•(m/q)a = E + v × B

•Key specifications are resolution, mass measurement accuracy, and sensitivity.

Mass analyzers separate ions based on their mass-to-charge ratio (m/z)

Time-of-flight (TOF) Mass Analyzer

+

+

+

+

Source Drift region (flight tube)

dete

ctor

V

• Ions are formed in pulses.

• The drift region is field free.

• Measures the time for ions to reach the detector.

• Small ions reach the detector before large ones.

Particles on which the same amount of work was done will have the same kinetic energy. Work is Force time distance Force = qE force is proportional to charge and field strength The distance is the same for all particles so the kinetic energy of all particles is the same and proportional to: ½ m/q v2

So a particle with twice the m/z will have a reduction in velocity of by a factor of the square root of 2. In general, the reduction in velocity is proportional to the square root of m/z. The consequence of this is that at higher m/z the smaller the difference in arrival times.

Time-of-flight (Tof) analyzer

Ions are accelerated so that they have equal kinetic energy. The ions “drift” down a 1 - 1.5 meter tube before striking a photomultiplier detector. “time of flight” (t) depends on the mass of the ion (m), where t = (m/2eV)1/2*D V is the applied potential and D is the flight tube distance. For a given instrument, the flight time varies as the square root of the mass of the ion.

Resolution 2 x 104

No upper limit of mass

Scan times ~ 1 µsec, good for LC-MSMS

Source

Accelerating pulse

Tof tube (1.0 - 1.5 m)

Effective flight tube (3.0 m)

Detector

Detector Reflector

Quadrupole Mass Analyzer Uses a combination of RF and DC voltages to operate as a mass filter.

• Has four parallel metal rods.

• Lets one mass pass through at a time.

• Can scan through all masses or sit at one fixed mass.

Quadrupole analyzer

Ions are accelerated electrically (5-15V) and passed along the long central axis of four rods arranged symmetrically. By applying combined DC and oscillating RF potentials, the ions drift along irregular flight paths along the rod axis. The DC/RF ratio is held constant and the absolute values of DC and RF are varied. Only ions with a particular m/z value have stable trajectories for a given value of DC and RF. If DC is set to 0, then all ions have stable trajectories. A scan can be accomplished over a period of 10-1000 msec.

Mass resolution 2 x 103

Tolerant of relatively high pressure (10-4 torr)

Upper limit for m/z is 3,000-4,000

Ion Traps

The ion trap is an energy well - ions with sufficient energy to enter the trap are retained by an energy barrier on the exit side of the trap. The advantage of the ion trap is that it accumulates selected ions prior to their analysis giving it high initial sensitivity (detection limit of approx. 20 fmol). Ions are fragmented by collision with helium gas and their daughter ions analyzed within the trap. Selected daughter ions can undergo further fragmentation, thus allowing MSn. The ion trap has a high efficiency of transfer of fragment ions to the next stage of fragmentation (unlike the triple quadrupole instrument).

Inlet Ion source

Mass Analyzer Detector

Data System

High Vacuum System

Microchannel Plate Electron Multiplier Mass analyzer/ion trap AC image

Detector

Multichannel Plate (MCP)

• High gain • High Temporal Resolution • High Spatial Resolution

Detecting in the ion trap Ion cyclotron resonance (ICR)

7 Tesla magnet, or 9.4 T or 12 T or 14.5 T

FT-Ion Cyclotron Resonance MS

ω = qB/m

Frequencies are converted to masses.

https://www.youtube.com/watch?v=7EHngA4S3Ws&feature=youtu.be&t=45

Orbitrap Mass Analyzer

Block Diagram of Triple Quad

Thermo-Finnigan LTQ-FT

Quadrupole – Time of Flight (Q-ToF)

Ion Mobility Mass Spec (IMS)

• Provides information about the size and shape of a molecule in the gas phase

• Imagine putting a size exclusion column in front of the mass spectrometer

Ion Mobility Mass Spec (IMS)

Provides information about the Size and shape of a molecule In the gas phase

Inlet Ion source

Mass Analyzer Detector

Data System

High Vacuum System

PC

Data System

The mass spectrum shows the results R

elat

ive

Abun

danc

e

Mass (m/z)

0

10000

20000

30000

40000

50000 100000 150000 200000

MH+

(M+2H)2+

(M+3H)3+

MALDI TOF spectrum of IgG

Assigning numerical value to the intrinsic property of “mass” is based on using carbon-12, 12C, as a reference point. One unit of mass is defined as a Dalton (Da). One Dalton is defined as 1/12 the mass of a single carbon-12 atom. Thus, one 12C atom has a mass of 12.0000 Da.

How is mass defined?

Isotopes

+Most elements have more than one stable isotope. For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da.

+Why do we care? Mass spectrometers can “see” isotope peaks if their resolution is high enough.

If an MS instrument has resolution high enough to resolve these isotopes, better mass accuracy is achieved.

Element Mass AbundanceH 1.0078

2.014199.985%0.015

C 12.000013.0034

98.891.11

N 14.003115.0001

99.640.36

O 15.994916.999117.9992

99.760.040.20

Stable isotopes of most abundant elements of peptides

Calculating Molecular Weight Monoisotopic mass (exact mass) Mass of an ion calculated using the exact mass of the most abundant isotope. Average mass Mass of an ion calculated using average atomic weight of each element. Nominal mass Mass of an ion calculated using the integer mass of the most abundant isotope.

Isotopic Distribution of Carbon

1981.84

1982.84

1983.84

Mass spectrum of peptide with 94 C-atoms (19 amino acid residues)

No 13C atoms (all 12C)

One 13C atom

Two 13C atoms

“Monoisotopic mass”

m/z

4360.45

4361.45

Isotope pattern for a larger peptide (207 C-atoms)

Mass spectrum of insulin

12C : 5730.61

13C

2 x 13C

Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used.

Monoisotopic mass

Monoisotopic masscorresponds tolowest mass peak

When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.

Average mass Average mass corresponds to the centroid of the unresolved peak cluster

When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass.

6130 6140 6150 6160 6170

Poorer resolution

Better resolution

What if the resolution is not so good? At lower resolution, the mass measured is the average mass.

Mass

Two peptides - same nominal mass - simulation

Peptide mixture: [Val5]-Angiotensin II Lys-des-Arg9-Bradykinin Sequence: DRVYVHPF KRPPGFSPF Formula: C49H69N13O12 C50H73N13O11

Exact mass: [M+2H]2+ = 516.76671 [M+2H]2+ = 516.78490 ∆m (mmu): 18.2 mmu

RP = 18,000

Inte

nsity

(%)

0

20

40

60

80

100

m/z 516.65 516.70 516.75 516.80 516.85 516.90

516.77581 (observed)

516.76671 (correct)

516.78490 (correct)

RP = 56,700

Inte

nsity

(%)

0

20

40

60

80

100

m/z 516.65 516.70 516.75 516.80 516.85 516.90

516.78490 516.76671

15.01500 15.01820 15.02140 15.02460 15.02780 15.03100Mass (m/z)

100

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

yISO:CH3

15.0229M

FWHM = ∆M

R = M/∆M

How is mass resolution calculated?

Mass measurement accuracy depends on resolution

0

2000

4000

6000

8000

Cou

nts

2840 2845 2850 2855

Mass (m/z)

Resolution = 14200

Resolution = 4500

Resolution =18100 15 ppm error

24 ppm error

55 ppm error

High resolution means better mass accuracy

489 490 491 492 493 494 495 496m/z

489 490 491 492 493 494 495 496m/z

489 490 491 492 493 494 495 496m/z

FT-ICR MS

RP = 491.2594 / 0.0055 amu = 89,319

Mass Resolution = m / Δm 50%

489 490 491 492 493 494 495 496m/z

LTQ MS

RP = 491.45 / 0.69 amu = 712

How Do We Determine Charge State?

489 490 491 492 493 494 495 496m/z

492.25

492.58 ∆ = 0.33

How Do We Determine Charge State of a Protein?

1599.8

1499.9 1714.1

1845.9 1411.9

1999.6 2181.6

m/z

m/z = (MW + nH+)/n

040308

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

Mix 406 (7.446) Cm (399:413) TOF MS ES+ 1.06e3985.7959949.3958

949.2087

915.3472883.8630

854.4720

826.8801

826.7056

1025.1672

1025.30191070.7888

1114.34631164.8209

1220.1954

040308

910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080 1090m/z0

100

%

Mix 406 (7.446) Cm (399:413) TOF MS ES+ 1.06e3985.7959949.3958

949.2087915.4742

915.3472

914.5703

913.5538

917.9626918.0758

918.3589

918.5995948.7340

919.1232

951.9443952.0451 985.5759

952.2180

952.4198

952.7657 985.2242953.0972

1025.1672988.4808

988.7010

988.7597

988.90661024.7339989.0682

989.4500 1024.6140

1025.30191067.8568

1028.08501067.6586

1028.2646

1028.30961028.4144

1028.6390 1067.3838

1070.7888

1070.9875

1071.0946

1071.3544

1071.5532

Mass Spectrum of Denatured Intact Protein

Q1

Ion Mirror (reflector)

Effective Flight Path = 2.5 m

Q2

Q0

Sample

Quadrupole-Time of Flight (Q-ToF)

Fragmentation of Peptides

87.04 57.04

Ion Mobility Mass Spec (IMS)

• Provides information about the size and shape of a molecule in the gas phase

• Imagine putting a size exclusion column in front of the mass spectrometer

Ion Mobility Mass Spec (IMS)

Provides information about the Size and shape of a molecule In the gas phase