BITS - Introduction to Mass Spec data generation

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BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011

Lennart Martens [email protected]

mass spectrometry basics

lennart martens

[email protected]

Department of Biochemistry, Ghent University Department of Medical Protein Research, VIB

Ghent, Belgium



AMINO ACIDS AND PROTEINS



From: http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-1/ch5-amino-acids.jpg

Amino Acids and their properties



NH2

NN

NN

NN

R1

O

H

R2

O

H

R3

O

H

R4

O

H

R5

O

H

R6

O

H

R7

OH

O

residue

amino terminus carboxyl

terminus

NC

C

R1

O

O

H

H

H

NC

C

R2

O

O

H

H

H

NC

C

R1

OH

H NC

C

R2

O

OH

H

amino group carboxyl group

side chain

+

peptide bond

OHH

+

A protein backbone



NH2

NN

NN

NN

R1

O

H

R2

O

H

R3

O

H

R4

O

H

R5

O

H

R6

O

H

R7

OH

O

Methionine Glycine Alanine Serine Tyrosine Leucine Arginine

Met Gly Ala Ser Tyr Leu Arg

M G A S Y L R

MGASYLR

A protein sequence



MASS SPECTROMETRY:

CONCEPTS AND COMPONENTS



sample ion source mass analyzer(s) detector digitizer

Generalized mass spectrometer

All mass analyzers operate on gas-phase ions using electromagnetic fields. Results are therefore plotted on a cartesian system with mass-over-charge (m/z) on the X-axis and ion intensity on the Y-axis. The latter can be in absolute or relative measurements. The ion source therefore makes sure that (part of) the sample molecules are ionized and brought into the gas phase. The detector is responsible for actually recording the

presence of ions. Time-of-flight analyzers also require a digitizer (ADC).

Schematic view of a generalized mass spec



laser irradiation

target surface

desorption proton transfer

analyte

matrix molecule

H+

+

Ion sources: MALDI

ν⋅h

+

+ +

+

+ +

+

+

+ +

+

+ + +

+

+

+ + + +

+ +

+

+ +

Gas phase

MALDI sources for proteomics typically rely on a pulsed nitrogen UV laser (υ = 337 nm) and produce singly charged peptide ions. Competitive ionisation occurs.

The term ‘MALDI’ was coined by Karas and Hillenkamp (Anal. Chem., 1985) and Koichi Tanaka received the 2002 Nobel Prize in Chemistry for demonstrating MALDI ionization of biological macromolecules (Rapid Commun. Mass Spectrom., 1988)

high vacuum

Matrix Assisted Laser Desorption and Ionization (MALDI)



Matrix properties:

• Small organic molecules • Co-crystallize with the analyte • Need to be soluble in solvents compatible with analyte • Absorb the laser wavelength • Cause co-desorption of the analyte upon laser irradiation • Promote analyte ionization

MALDI matrix molecules



matrix sample matrix + sample

+

spot on target and let air-dry

matrix crystals

1-2 mm

stainless steel, gold teflon, …

MALDI – spotting on target



0

+ 0 +

0 0 0 0

0 0

0

0

0

+

+

+ +

+

+

+

+

+ + +

+ + +

+

+ +

+ +

+ +

needle

barrier

+

3-5 kV

droplet evaporation and charge-driven fission

or ion expulsion

m/z analyzer inlet

0

0

0

0

0

0

0

0

0 0

0

0

evaporation only

John B. Fenn received the 2002 Nobel Prize in Chemistry for demonstrating ESI ionization of biological macromolecules (Science, 1989) – ESI is also used in fine control thrusters on satellites and interstellar probes…

ESI sources typically heat the needle to 40°to 100°to facilitate nebulisation and evaporation, and typically produce multiply charged peptide ions (2+, 3+, 4+)

nebulisation

www.sitemaker.umich.edu/mass-spectrometry/sample_preparation

N2

N2 sample

Ion sources: ESI

Electospray ionization (ESI)



A B

solvent mixer

(100–5 µm) needle

(nano) RP column (nano) spray

aqueous solvent H2O + 0.1% FA + 2–5% ACN

organic solvent ACN + 0.1% FA

Nanospray ESI sources (5-10 µm diameter needle) achieve a higher sensitivity, probably due to the higher surface-to-volume ratio. For a spherical droplet this ratio is:

ESI – online LC and solvents

34

3rV ⋅=

π24 rA ⋅⋅= π

DrVA 63

==



source

extraction plate (30 kV)

detector field-free tube (time-of-flight tube)

> 1 meter

high vacuum

Analyzers: time-of-flight (TOF)

VqEk ⋅=mEvvmE k

k⋅

=↔⋅

=2

2

2

2

2

22

222x

tV

txV

vV

qm ⋅⋅

=

⋅=

⋅=

sample ions

We can now relate m/q (or the more commonly used m/z) to the velocity of the ion, and using Newton’s kinematica we can relate the speed to the travel time and (known + exactly calibrated) field-free tube length



Ekin > Ekin

ion mirror reflectron

detector

with reflectron

Analyzers: time-of-flight – reflectron




source

time t0 tx tx + 100 ns


source


source

field gradient **** *** ** *

Analyzers: time-of-flight: delayed extraction



Resolution in mass spectrometry is usually defined as the width of a peak at a given height (there is an alternative definition based on percent valley height). This width can be recorded at different heights, but is most often recorded at 50% peak height (FWHM).

From: Eidhammer, Flikka, Martens, Mikalsen – Wiley 2007

average mass

monoisotopic mass

Resolution and why it matters



ring electrode

capping electrode

capping electrode

source detector

Wolfgang Paul and Hans Georg Dehmelt received the 1989 Nobel Prize in Physics for the development of the ion trap.

Ion traps operate by effectively trapping the ions in an oscillating electrical field. Mass separation is achieved by tuning the oscillating fields to eject only ions of a specific

mass. Big advantages are the ‘archiving’ during the analysis, allowing MSn.

DC/ACRF voltage

Analyzers: ion trap (IT)



Quadrupole mass analyzers also use a combined RF AC and DC current. They thus create a high-pass mass filter between the first two rods, and a low-pass mass filter

between the other two rods. The net result is a filter that can be fine-tuned to overlap (and thus permit) only in a specific m/z interval; ions of all other m/z values will be ejected.

permitted m/z

ejected m/z

ejected m/z

)cos( tVU ω⋅+−

)cos( tVU ω⋅++

Analyzers: quadrupole (Q)



Different variations of electron multiplier (EM) detectors are in use, and they are the most common type of detector. An EM relies on several Faraday cup dynodes

with increasing charges to produce an electron cascade based on a few incident ions.

single ion in

106 electrons out

20V

60V

100V

40V

80V

120V

Detectors: electron multiplier



An FT-ICR is essentially a cyclotron, a type of particle accelerator in which electrons are captured in orbits by a very strong magnetic field, while being accelerated by an

applied voltage. The cyclotron frequency is then related to the m/z. Since many ions are detected simultaneously, a complex superposition of sine waves is obtained. A Fourier

transformation is therefore required to tease out the individual ion frequencies.

electrodes

strong magnetic field

ion orbit

Fourier transform ion cyclotron resonance (FTICR)



An OrbiTrap is a special type of trap that consists of an outer and inner coaxial electrode, which generate an electrostatic field in which the ions form an orbitally harmonic oscillation along the axis of the field. The frequency of the oscillation is inversely proportional to the

m/z, and can again be calculated by Fourier transform. The OrbiTrap delivers near-FT-ICR performance, but is cheaper, much more robust, and

much simpler in maintenance. It is a recent design, only a few years old.

From: http://www.univ-lille1.fr/master-proteomique/proteowiki/index.php/Orbitrappe

Orbitrap



TANDEM MASS SPECTROMETRY

(TANDEM-MS, MS/MS, MS2)



source detector

Tandem-MS is accomplished by using two mass analyzers in series (tandem) (note that a single ion trap can also perform tandem-MS). The first mass analyzer

performs the function of ion selector, by selectively allowing only ions of a given m/z to pass through. The second mass analyzer is situated after fragmentation is triggered

(see next slides) and is used in its normal capacity as a mass analyzer for the fragments.

ion selector fragment mass analyzer

fragmentation

Tandem-MS: the concept



NH2 C H

CO COOH N H

CH2 R1

R2

C H

CO N H

C H

CO N H

C H

CH2

R3

R4

x3 y3 z3 x2 y2 z2 x1 y1 z1

a1 b1 c1 a2 b2 c2 a3 b3 c3

peptide structure

There are several other ion types that can be annotated, as well as ‘internal fragments’. The latter are fragments that no longer contain an intact

terminus. These are harder to use for ‘ladder sequencing’, but can still be interpreted.

This nomenclature was coined by Roepstorff and Fohlmann (Biomed. Mass Spec., 1984) and Klaus Biemann (Biomed. Environ. Mass Spec., 1988) and is commonly referred to as ‘Biemann nomenclature’. Note the link with the Roman alphabet.

Why tandem-MS?



This fragmentation method is called post-source decay (PSD) and relies on a single unimolecular event, in which a highly energetic (metastable)

ion spontaneously fragments. PSD typically causes backbone fragmentation. y and b ions are by far the most prevalent fragment types, although TOF-TOF instruments (see later) specifically also yield substantial internal fragments.

) ) ( ( activated ion (metastable)

non-activated ion (stable)

precursor ion

fragment ions

200 400 600 800 1000 1200 1400 1600 m/z

laser

Creating fragments (unimolecular)



This fragmentation method is called collision-induced dissociation (CID) and relies on a series of bimolecular events (collisions) to provide the peptide precursor with

sufficient energy to fragment. CID typically causes backbone fragmentation. y and b ions are by far the most prevalent fragment types.

The collision gas is typically an inert noble gas (e.g.: Ar, He, Xe), or N2.

collision cell

∆V

gas inlet

collision gas (atom or molecule)

selected peptide

Creating fragments (bimolecular I)



This fragmentation method is called electron-capture dissociation (ECD) or electron-transfer dissociation (ETD) and relies on a single impact of an electron on a peptide

precursor. This high-speed impact immediately imparts sufficient energy to fragment the precursor (non-ergodic process). Like CID, ETD and ECD typically cause backbone

fragmentation, but they typically result in c and z ions. ECD is only workable in FT-ICR mass spectrometers, whereas ETD is used in traps.

fragmentation cell

∆V

electron source

electron

selected peptide

- -

- - -

-

Creating fragments (bimolecular II)



A CID FRAGMENTATION PRIMER



peptide X

peptide H+

0

2

4

6

8

10

12

14

Alanine

Arginin

e

Aspara

gine

Aspart

ic Acid

Cystei

ne

Glutam

ic Acid

Glutam

ine

Glycine

Histidi

ne

Isoleu

cine

Leuc

ineLy

sine

Methion

ine

Pheny

lalan

ine

Proline

Serine

Threon

ine

Tryptop

han

Tyrosin

eVali

ne

NH2–

–COOH

R–group

pKa

H+

fragmentation event

Assumption

Hypothesis

pKa values

CID fragmentation: the mobile proton model



From: Wysocki et al, J. Mass Spectrom., 2000

The necessary collision energy to achieve a given fragmentation efficiency is highest and equal for both 1+ peptides on the left (the R present in both sequesters the charge), and is higher for PPGFSPFR for the 2+ peptides. This is because the latter has an N-terminal P, which has a relatively high pKa for the N-terminus; so the second available charge is located at either end of the peptide. The other peptide has no real main contender (in pKa) for the second charge, and therefore fragments more easily. On the right, having two R’s in the sequence negates the effect of double charge (both charges are sequestered by an R).

Illustration of the MPM hypothesis



peptide 1+

fragmentation

1+ ?

b-ion y-ion

peptide 2+

fragmentation

b-ion y-ion

1+ 1+

Ionization and fragmentation



A SPECIAL FLAVOUR OF MS/MS:

MULTIPLE REACTION MONITORING



mass filter 1 mass filter 2 collision cell

selected peptides

fragments of both peptides

selected fragment

MRM removes noise, yielding better signal-to-noise ratio MRM removes ‘contaminating’ peaks, aiding targeted identification

MRM works well with proteotypic peptides MRM can be performed with Q-Q-Q, Q-LIT and IT instruments

peptide mixture

Multiple Reaction Monitoring (MRM)



MS1 analysis less complex peptide fractions

MS2 analysis

fragment selection

Multiple Reaction Monitoring is the most often used name for this method, even though the IUPAC draft standard for mass spectrometry does not endorse this term, and prefers Single Reaction Monitoring (SRM) instead. You can find both terms in use.

MRM analysis



MASS SPECTROMETER

CONFIGURATIONS



ESI source

detector ion trap

Very simple and reliable instrument, that can perform MS and CID MS/MS thanks to the ion trap. Mass accuracy is relatively poor however, and the resolution is lacking as well (unable to distinguish isotopes, hampering charge state determination).

ESI ion trap



ESI source

detector quadrupole 1

Simple instrument, that recently attracted attention because it is well-suited for Multiple Reaction Monitoring (MRM). It can perform MS and MS/MS, where the

first quadrupole is the ion selector, the second quadrupole a collision cell and the third quadrupole a mass analyzer. Due to the ‘wastefulness’ of a quadrupole as mass

analyzer, it is not very popular for general MS/MS analysis, however.

quadrupole 2 quadrupole 3

ESI triple quadrupole



MALDI source

linear detector TOF tube

delayed extraction

reflectron

Relatively simple instrument, that can measure intact masses quite accurately and can perform fragmentation analysis through PSD (unimolecular fragmentation), but the yield of fragmentation is poor. Cheap and reliable, but out of date nowadays.

voltage gate for precursor

selection

reflectron detector

MALDI DE RE-TOF



MALDI source

linear detector TOF 1

delayed extraction

A modern version of the MALDI DE RE-TOF, the TOF-TOF relies on two TOF tubes in tandem. The second TOF is fitted with a reflectron. Mass accuracy and resolution are very high and the instrument can perform MS and MS/MS, both for peptides as well as

whole proteins. The archiving nature of the MALDI targets allows the instruments to scan a single sample more thoroughly. TOF-TOF instruments are also well-suited for profiling.

voltage gate for precursor

selection

reflectron

reflectron detector

TOF 2

source 2

MALDI TOF-TOF



ESI source

detector

quadrupole

One of the first hybrid mass spectrometers, the Q-TOF combines the excellent precursor selection characteristics of the quadrupole with the mass resolution and accuracy of the

TOF tube. The ESI source produces multiply charged ions, and can be coupled to an online (nano-) LC separation. A Q-TOF is a very good instrument when high-quality data are more important than high-throughput, and is well suited for protein characterisation.

collision cell reflectron

TOF

pusher

ESI quadrupole time-of-flight (Q-TOF)



quadrupole capping electrode

capping electrode

source detector

Linear ion traps store ions in a fixed linear trajectory rather than in a ‘blob’. The major advantage is the increased capacity for trapping ions. This allows for

a broader dynamic range and better quantitation performance. The linear ion trap is these days often encountered as the workhorse mass spectrometer in many labs, and can be extended with the highly accurate orbitrap or FT-ICR mass analyzers/detectors.

ESI quadrupole linear ion trap



ESI source

linear ion trap

C-trap

FT-ICR Orbitrap

The combination of a linear ion trap and a high-resolution, high-accuracy FT analyzer allows for a broad dynamic range and highly accurate mass measurements.

Since the ion trap can be used as a collision cell, the FT analyzer can also measure the resulting fragments with high accuracy.

ESI linear ion trap FT-ICR or Orbitrap



Thank you!

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BITS - Introduction to Mass Spec data generation