Tandem Mass Spectrometry

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Section 5 Tandem Mass Spectrometry

• Part 1
• Fragmentation
• MS/MS Analyzers
• MS/MS Data

Part 2
Protein Identification in Large-scale Proteomics
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(No Transcript)
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Basics of MS/MS Analysis

• Motivation
• Often one or two peptides whose amino acid
  sequence is know can identify the protein
• to which they belong (this is not true if we
  only the mass of the peptides as is the case in
  MS
• The second step in MS/Ms is designed to provide
  some information of the amino acid
• sequence of a peptide
• if we know only the mass of a peptide, plus that
  it contains a modification, it is impossible
• to locate the exact site of the modification
  but we obtain information for each amino acid,
• then we may be able to infer the modification
  site
• Two mass analyzers
• They may be one analyzer operating in two
  different modes
• The first analyzer selects peptides in a
  particular range of m/z (typically these are
  peptides
• with m/zs centered around the m/z of a
  specific peptide
• the selected peptides are fragmented, and the
  second analyzer measures the m/z of the
• charged fragments
• Terminology
• peptide ion precursor ion parent ion
  charged peptide chosen for
• 
  fragmentation
• fragment ion product ion daughter ion a
  charged fragment produced

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Fragmentation
1. Fragment types

• (a) Backbone fragments
• Main backbone fragments a-, b-, c-, x-, y-, z-
  fragments

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Fragmentation -- continued
1. Fragment types -- continued

• (a) Backbone fragments continued
• partial or total fragmentation of side chains

The main backbone fragments (a-, b-, c-, x-,
y-, z-) may undergone partial or complete
fragmentation in their side chains, yielding new
types of fragments. The most common are -
d-fragments generated from a-fragments by
partial fragmentation of a side chain
- v-fragments generated from y-fragments by
total fragmentation of a side chain -
w-fragments generated from z-ions by partial
fragmentation of a side chain (the
side chains that are fragmented are typically the
side chains of amino acids where
the fragmentation produces the main backbone
fragments
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Fragmentation -- continued
1. Fragment types -- continued
(b) Immoniums an internal fragment composed of
a single amino acid
formed by a combination
of an a- fragment and a y-fragment
but subsequently, the fragments
captures a proton H
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Fragmentation -- continued
1. Fragment types -- continued
(c) internal fragments - normally generated
by b- and y-type fragmentations - typically
they short having 5 or less amino acids NOTE
immoniums is a special internal fragment
(d) Losses water loss (H2O, mass 18.011
Da) commonly occurs by partial
fragmentation of the side chain of
S (serine), T (threonine), D (asparatic acid), E
(glutamic acid) ammonia loss (NH3, mass
17.027 Da) commonly occurs by
partial fragmentation of side chains of
R (argine), K (lysine), N (asparagine), Q
(glutamine
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Fragmentation -- continued
2. Fragmentation techniques

• Fragmentation is central in the interpretation of
  MS/MS spectra. Its
• chemistry is not well understood. Its role in the
  protein identification
• problem and the interpretation of the data is
  much more subtle and
• complex than that of digestion and modifications
  (and noise).
• Fragmentation vs Ionization
• - fragmentation occurs when excess internal
  energy is built in the bonds of an ion the
  excess
• energy drives certain reactions that brake
  the bonds to generate ions and neutral species
• - ions formed in any ionization process
  (including ESI and MALDI), can be classified
  according
• their stability during the mass
  measurement process as stable, unstable, and
  metastable ions. The
• majority of the ions produced by MALDI and
  ESI are stable
• -- stable ions remain intact and do not
  fragment because they have acquired insufficient
  energy
• during ionization (soft ionization
  sources)
• -- unstable ions have sufficient
  internal energy to fragment while still in the
  ionization source (this
• occurs especially, if the ionization
  source uses higher energies)
• -- metastable ions have an intermidiate
  amount of excess energy, and fragment after
  moving
• from the ion source into the mass
  analyzer as a result they are not seen in the
  mass spectrum,
• unless specific adjustments are made
  in m/z analysis

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Fragmentation -- continued
2. Fragmentation techniques -- continues

• General Division of Fragmentation Techniques
  (according to the location of fragmentation
  relative to the ion source)
• In-Source Decay (ISD)
• -- fragmentation occurs simultaneously
  with ionization
• -- this is useful only for small
  molecules, but not suitable for peptides (or
  proteins)
• Post-Source Decay (PSD)
• - fragmentation occurs after the ion source
• - but a PSD fragmentation still could be
  before the parent ion is observed also PSD
• may refer to fragmentation of metastable
  ions
• - In proteomics, fragmentation procedure are
  PSD procedures that are designed to
• fragments charged peptides after the m/z
  of the peptide ion has been detected or selected

• Major peptide fragmentation procedures
• CID (Collision Induced Dissociation also
  called CAD Collision Activated Dissociation)
• most common technique in proteomics
• ECD ( Electro Capture Dissociation)
• ETD ( Electron Transfer Dissociation)
• LID ( Laser Induced Dissociation)

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Fragmentation -- continued
2. Fragmentation techniques -- continued
Major peptide fragmentation procedures CID
(Collision Induced Dissociation also called CAD
Collision Activated Dissociation)

• most commonly used in proteomics
• it generates mostly y- and b-ions
• usually combined with ESI, but sometimes also
  with MALDI
• collision cell
• - this is a special chamber in the spectrometer
  where
• fragmentation occurs
• - the cell is a high-pressure region that
  contains a neutral
• collision gas (typically argon)
• - As the charged peptide collides with the gas
  molecules,
• a portion of its kinetic energy is converted
  into internal
• potential energy that makes the peptide
  unstable and drives
• fragmentation reactions that occur inside the
  cell
• - CID fragmentation produces both charged
  fragments and neutral losses
• - what charged fragments and neutral losses CID
  produces depends on the peptide
• on the energy of the gas
• - in proteomics, the energy used is lower than
  100ev (typically 25-70 eV)
• - energies larger than 100 eV can fragment
  higher mass peptides, but it produces many more
• fragments, there making the interpretation
  of the spectrum more difficult

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Fragmentation -- continued
2. Fragmentation techniques -- continued

• Major peptide fragmentation procedures --
  continued
• ECD (Electron Capture Dissociation)
• It produces mainly c- and z-ions, and
  occasionally a-ions
• It is less efficient than CID, and it requires
  special requires special spectrometer (FT-ICR)
• For these reasons it is used in special problems
• fragmentation occurs by
• - charged peptide with more than one positive
  charges are trapped in a bath of low-energy
  electrons
• - the peptide captures such electrons, and
  fragments quickly
• - the more charges the peptide has, the more
  electrons it captures, resulting to faster
  fragmentation
• - it can fragment large multiply charged
  peptides (and even intact proteins)
• ETD (Electron Transfer Dissociation)
• It replaces the free electrons of ECD by anions
  (negatively charged ions)
• The anions transfer electrons to the peptide,
  inducing fragmentation
• Because it is easier to trap large anions long
  enough, ETD does not require FT-ICR
• LID (Laser Induced Dissociation)
• It uses a laser in fact the laser used in MALDI
  can be used here, but inside the mass
  spectrometer
• and not at the ion source
• LID produces primarily a-, b-, and y-ions

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Fragmentation -- continued
3. The Mobile Proton Theory of Fragmentation

• Tryptic peptides nearly always have an R
  (argine) or K (lysine) at their C-terminus
• but they may have interior R and/or K due to
  cleavage missing or presence of P (proline), or
• presence of pairs KR or RK
• Also tryptic peptides may contain H (histidine)
  amino acids H is basic
• - K, R, H, and the N-terminus are basic amino
  acids, and therefore accept protons during
  ionization
• - Also, other less basic amino acids may
  capture protons during ionization
• - in fact even the acidic amino acids E
  (gluatmic acid) and D (aspartic acid) may host
• protons, albeit weakly
• MALDI typically generates single charged
  peptides, and the single charge is hosted by and
  large by
• the C-terminal R or K (although, sometimes it
  may be located at the N-terminus group
• - ESI generates most peptides with charge 2, 3,
  4 (rarely higher), although doubly charged
  peptides
• constitute the majority
• - the charges are hosted primarily by the basic
  amino acids (K, R, H) and the N-terminus, but
• sometimes by other amino acids
• - for example, if a peptide has charge 3 and
  only one interior basic amino acid (say, H). Then
• most likely on charge is located at the
  C-terminal R or K, another is located at H, and
  the third
• at the N-terminus group
• - If a charge is hosted by one of the basic
  amino acids (R, K, H), then it basically
  sequestered by these
• amino acids (nearly 100 in the case of R).

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Fragmentation -- continued
3. The Mobile Proton Theory of Fragmentation --
continued
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The 20 amino acids Standard grouping not
showing, e.g. the degree of basicity, Acidicity,
etc
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The 20 amino acids and their properties
pI is related to the how much basic an amino acid
is the higher the pI the more strongly basic the
amino acid is
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Fragmentation -- continued
3. The Mobile Proton Theory of Fragmentation --
continued
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Fragmentation -- continued
3. The Mobile Proton Theory of Fragmentation --
continued

• - fragmentation occurs at the site where the
  mobile proton moves to
• - without the mobile proton only limited
  fragmentation is observed with even the most
  severe
• low-energy collision conditions
• - For this reason, multiply charged peptides
  (such as those produced by ESI) are subjected to
  more
• informative fragmentation
• - Internal charges of tryptic peptides (hosted
  by basic amino acids), have an effect on the
  place where
• a mobile proton moves to
• - typically the mobile proton does not move to
  peptide bond near the internal charge. Hence the
• fragmentation pattern is altered by an
  internal charge
• Fragmentation induced by mobile protons is
  called charge-directed fragmentation
• - Charge-directed fragmentation of a peptide
  bond may occur through a number of pathways
• - at low-energy collisions the main pathways
  are shown in the next slide
• - The fragmentation pathway proceeds through
  cyclic intermediate that subsequently fragments
  by
• one of two reactions Reaction 1 favors
  b-ions, while Reaction 2 favors y-ions

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Fragmentation -- continued
3. The Mobile Proton Theory of Fragmentation
continued
Two major fragmentation pathways
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Fragmentation -- continued
3. The Mobile Proton Theory of Fragmentation --
continued

• Charge-directed fragmentation
• fragmentation of a peptide bond may occur
  through a number of pathways
• at low-energy collisions the main pathways
  proceed through cyclic intermediate that
  subsequently
• fragments by one of two reactions Reaction 1
  favors b-ions, while Reaction 2 favors y-ions
• - For a singly charged peptide protonated at the
  N-terminus, b-ions dominate
• - but for doubly charged peptides with one
  charge at the N-terminus and the other charge at
  the
• C-terminal R or K, y-ions dominate although
  Reaction 1 is more dominant than Reaction 2. The
• reason for this is the y-fragment is always
  charged also, the N-terminus mobile charge
• has a
  possibility that it is transferred to the y-ion
  by Reaction 2
• Charge-Remote Fragmentation
• This is another mechanism for fragmentation
  (even for singly charged peptides)
• - loosely bound protons to non-basic amino acids
  (such as acidic amino acids) can initiate
• fragmentation at the amide bond C-terminal
  to the acidic residue
• - this occurs for the acidic side chains of E
  (glutamic acid) and D (aspartic acid)
• Peptide Classes
• mobile peptides peptides whose number of basic
  residues (R, K, H) is less than number of charges
• non-mobile peptides peptides whose Rs is
  larger than the number of charges
• partially mobile peptides peptides that are
  neither mobile nor non-mobile

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Fragmentation -- continued
4. Statistics
E.A. Kapp et.al. Analytical Chemistry (2003)
75, 5251-6264
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Fragmentation -- continued
4. Statistics
Huang et.al AnalyticalChemistry (2005) 77,
5800-5813
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MS/MS Analyzers

• Basic Classification of MS/MS Analyzers
• MS/MS analyzers must perform two tasks
• - measure the m/z of a peptide or select a
  small range of ratios around an m/z value
• - measure the m/z values of the ion-fragments
• The two tasks are either done by two analyzers
  operating before and after fragmentation
• or by a single analyzer that functions in two
  different modes and also encompasses the
• fragmentation mechanism

MS/MS analyzers

• The two types of analyzers are called
• in-space analyzers and in-time
  analyzers

One device working in two different
modes Full-scan mode peptide ions are
analyzed and retained, and an MS spectrum is
recorded MS/MS mode - analyzer retains only
ions that fall within an m/z region, ejecting
or cutting-off peptide ions not in this range
- then the retained ions are subjected to
fragmentation - the m/z of the fragments is
measured by the analyzer operating in the
full scan mode The analyzer is configured to
switch contineously between the two modes
Two analyzers, one for selecting the m/z
range, and the other for measuring the m/z of the
fragments between them lies the cell
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MS/MS Analyzers -- continued

• Reflectron-TOF
• The part of the instrument that allows ion
  selection
• is the ion gate ( mass gate timed ion
  selector TIS)
• An m/z range is selected by switching off a
  voltage on the
• ion gate when the peptides of interest (i.e
  peptides in the
• range) pass through
• Peptide ions outside the range arrive before or
  after the
• voltage is switched off and therefore are
  deflected by the
• electric field, away from the entrance to the
  flight tube
• The fragmentation is that of metastable peptides
  and occurs in
• the field free region
• The velocity of the fragments is the same as
  that of the precursor
• peptides
• for this reason the instrument is fitted with a
  reflectron that
• reflects ion according to m/z
• The larger the m/z the deeper it penetrates the
  reflectron
• sometimes unwanted ions can form at the
  reflectron

A variant of the Reflectron-TOF in which
fragmentation occurs before the ion gate
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MS/MS Analyzers -- continued
quadrupole

• Triple-Quadrupole (TQ)
• TQ is typically combined with ESI
• It consists of three Quadrupoles
• Each Q has four rodes arranged in pairs
• and subjected to a combination of a
• DC (direct current) and RF (radio frequency)
• alternating voltage
• Ions passing through the field created by the DC
• and RF voltages follows a spiral trajectory
• The radius of the ions trajectory depends on
  the
• ions m/z
• For a specific voltage, only ions with certain
  m/z
• will reach the detector, while the others will
• collide with the rods or ejected from the Q
• by scanning over the voltage and observing
• the ion abundances for each voltage interval,
• a mass spectrum is constructed
• In TQ, the middle quadrupole is used as for
• fragmentation (usually CID), while the first

(a) Analysis in MS/MS mode
triple Quadrupole
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MS/MS Analyzers -- continued

• In Trap (IT)
• This is an in-time analyzer
• the ions (peptides or fragments)
• are trapped in a confined space
• Three types - 3D-IT
• - Linear Ion Trap (LIT)
• - Orbitrap
• 3D-IT
• has three electrodes (two cap electrodes and one
  ring between the caps
• It is similar to a Q two of the rods form the
  end caps , a third forms the
• ring electrode, and a fourth collapsing to a
  point at the center of the ring
• An AC voltage is applied at the end caps and an
  RF voltage between the ring and the caps
• An m/z range is selected and all other peptide
  ions are ejected by adjustin the voltage
• collision gas is introduced into the trap, while
  the voltages are adjusted
• to increase the kinetic energy of the
  remaining peptides, thereby fragmenting them
• the fragment ions are ejected from the trap, and
  detected by a detector
• The size of the trap is equal to a lemon this
  is relatively small and limits the dynamic range
  of 3D-IT
• This disadvantage is corrected in LIT and
  Orbitrap

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MS/MS Analyzers -- continued

• In Trap (IT)
• Orbitrap
• It consists of an outer and an inner electrodes
  that
• generate an electric field with electrstatic
  potentiale

• The ions perform a harmonic oscillation along
  the z-axis
• The frequency of the ion oscillation along the
  z-axis is

• The frequency can be computed by FFT
• Orbitrap has high resolution and accuracy

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MS/MS Analyzers -- continued

• FT-ICR ( Fourier Transform Ion Cyclotron
  Resonance)
• Combining Analyzers
• Q-TOF
• Q-TRAP
• TOF-TRAP
• LIT-Orbitrap

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MS/MS Data
Normally MS/MS spectra are presented as MS and
MS/MS peaks

• - An MS/MS instrument outputs both the MS data
  (at least conceptually) as well as the MS/MS
• fragment data for each peptide
• - normally the MS data are presented after
  pre-processing for example, isotope peaks are
  replaced
• by one peak line (monoisotoping or
  deisotoping)
• - isotopic peaks, if available, can be used to
  determine the charge of the peptide (the peptide
  in the inlet
• above has charge 2)
• But LC-MS/MS instruments
• - often may not resolve the isotopic peaks
  hence the charge of the peptide has to be
  computed in
• a different way
• - also, the instrument selects a range of
  peptide around a pre-selected value for m/z
• - in fact the story is a bit subtler, as we
  will see next

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MS/MS Data -- continued
LC-ESI-MS/MS data

• LC chromatography (e.g. RP or SCX) separates
  peptides into bands (via hydrphobicity, charge,
  etc)
• Bands are sequentially eluted into ESI and then
  sputtered into the mass spectrometer
• - In MS instrument, the analyzer records the m/z
  value for each peptide separetely and constructs
• the m/z spectrum
• - but many MS/MS instruments, the MS spectrum
  is only conceptually constructed for example,
• the quadrupole and ion trap selectors select
  a range of m/z values around a preselected m/a
  value
• How do we separate peptides that may be in the
  same range?

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MS/MS Data -- continued
LC-ESI-MS/MS data

• As before, in an ESI-MS/MS instrument, the MS
• spectrum is first constructed at the time point
• at which the sample is infused into the MS/MS
• device., i.e. the spectrum is constructed for
• each scan infused into the instrument
• (in MALDI, this spectrum corresponds to a single
  spot)
• This MS spectrum contains multiple peaks
• corresponding to multiple peptides.
• Then some of these peaks are selected
  automatically
• selected for fragmentation
• The user can specify a set of selection
  criteria, e.g.
• - The n most intense peaks (typically n 3-8)
• - an inclusion list of specified m/z values
  (this may be
• useful in targeted applications)
• - exclusion of certain peaks that are suspected
  to come from contaminants such as keratin
• - the charge of the peptide (this assumes that
  the instrument has sufficient resolution to
  distinguish
• the isotopic peaks, which then can be used to
  find the charge z)
• The final MS/MS spectrum for each peptide is
  constructed from the accumulated spectra from
• scans (some instruments allow the user to
  specify the accumulation time)

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MS/MS Data
Peptide Charge Estimation from MS/MS Data

• There are various situations in which the charge
  of a peptide need to be estimated from MS/MS data
• (i.e. from the fragment spectrum of a
  peptide)
• (i) The MS spectra may be of low resolution (not
  unlikely for some MS/MS instruments) so that
• the isotopic peaks cannot be resolve or they
  may overlap of one peptides isotopic peaks with
• those of another peptide (in which case an
  estimated charge may not be accurate)
• (ii) - In triple quadrupole and ion trap
  analyzers (as in most MS/MS instruments), the
  system selects
• a small range of m/z values about a
  pre-selected m/z value. The peptides have
  narrowly spaced
• m/z values, as a consequence of which the
  signal to noise ratio is small this makes
  interpretation
• of the MS/MS data difficult in addition, it
  degrades the estimation of the charges of the
  peptides,
• for example when the isotopic peaks are
  incorrectly shifted (see next slide)
• The above related difficulties may lead to an
  non-accurate estimation of the peptide charge.
• This estimate can be improved by an analysis
  of peptides MS/MS data.
• This analysis is based on precise relation of
  the between the m/z value for the peptide and
• the m/z values of its complementary fragments
  (i.e. b-ions/y-ion, a-ions/x-ions, c-ion/z-ions

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MS/MS Data
Peptide Charge Estimation from MS/MS Data
Isotopic peaks may be degraded
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MS/MS Data -- continued
Peptide Charge from MS/MS Data -- continued
Masses of amino acids, proteins, peptides, and
fragments
amino acid
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MS/MS Data -- continued
Peptide Charge from MS/MS Data -- continued
Masses of amino acids, proteins, peptides, and
fragments-- continued
mass of a neutral protein or peptide sum of its
amino acids masses H HO
sum of
its amino acids masses 18
mass of H 1 Da Mass of HO 17 Da
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MS/MS Data -- continued
Peptide Charge from MS/MS Data -- continued
neutral peptide
Masses of amino acids, proteins, peptides and
fragments-- continued
The six main backbone fragments with z 1

• The y-ion and the c-ion get two extra protons
  (H)
• The indicated charge location is consistent with
  the proton mobile theory (when the peptide has
• a mobile proton at its N-terminus, and the
  proton can move at any amide oxygen or nitrogen)
• But the charges can be at any location

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MS/MS Data -- continued
Peptide Charge from MS/MS Data -- continued
neutral peptide
Masses of amino acids, proteins, peptides and
fragments-- continued

• Let p be a peptide and define
• Rp sum of masses of the peptides amino acids
• Mp RpHOH mass of the neutral peptide p
• Rii sum of masses of the amino acids of
  fragment i a, b, c, x, y, z
• Mi mass of singly charged fragment i a, b,
  c, x, y, z
• Then

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MS/MS Data -- continued
Peptide Charge from MS/MS Data -- continued
neutral peptide
Masses of amino acids, proteins, peptides and
fragments-- continued

• Let p be a peptide and define
• Rp sum of masses of the peptides amino acids
• Mp RpHOH mass of the neutral peptide p
• Rii sum of masses of the amino acids of
  fragment i a, b, c, x, y, z
• Mi mass of singly charged fragment i a, b,
  c, x, y, z
• Then

Noting that
one gets
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MS/MS Data -- continued
Peptide Charge estimation from MS/MS Data --
continued
neutral peptide
Method 1
Case 1 zp 1 or zp 1 If zp, then all
fragments will have z1. Therefore all fragment
peaks will be located before the peak for the
peptide. Thus if most of the peaks are before the
peak of the peptide the charge of the peptide
will be 1 Case 2 zp vs zp 3 Assume (i) No
neutral losses occur, (ii) if the peptide has
charge 3, no internal fragments occur
(iii) only b-ions and y-ions occur Let zb, zy
denote the charges of the b-ions and they-ions.
Using one
shows

• Consider now all pairs of peaks
• and record the number N1 of
• peaks that satisfy (), and the
• number of peaks N2 that satisfy
• () and ().
• If N1gt N2 then most likely zp2
• If N1lt N2 then most likely zp3

Which implies

• If zp2 then

()

• If zp3 then

()
()
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MS/MS Data -- continued
Peptide Charge estimation from MS/MS Data --
continued
Method 2

• If zp2 then

If zp2 then
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Observed Spectra

• The spectra cannot determine the peptide
• You need some sort of regularization use
• of a priori knowledge i.e. use of databases
• Fragmentation model