1. Analysis of MS Fragmentation

2. 2D GE and MS

3. Overview of Mass Spectrometry
Mass Spectrum
Mass Analyzer
Ionization M+/FragmentationSample Molecule (M)
Protonation : M + H+
MH
Cationization : M + Cat+
MCat+
Deprotonation: MH M-
+ H+
Electron Ejection: M M+.
+ e-
Electron Capture: M + e- M-.
Mechanism of Ionization

4. 1. Sample is
mixed in
matrix and
dried on
target.
2.Target is introduced
into high vacuum of
MS.
3.Sample is irradiated
with laser desorbing
ions into the gas phase
and the clock
measuring the time of
flight starts.
4.Ions are accelerated by an electric
field to the same kinetic energy and
they drift down the field free flight
tube where they are separated in
space
5.Ions strike the
detector at different
times depending on
the mass to charge
ratio of the ions
6.A data system controls all
the parameters, acquires the
signal vs. time and permits
data processing
Schematic of a MALDI-TOF Experiment

5. Schematic representation of a triple quadrupole MS
instrument
Quadrupole mass analyzer Trajectory of an ion
Operation in full scan
mode
Operation in MS/MS
mode

6. Schematic of mass spectrometer

7. CID
 CID is divided into low energy (<100 eV) and high
energy(>1000 eV) based on the collision energy.
 High energy CID produces more fragments, but is more
complicated to interpret.
 Low energy CID has a limit on the m/z it can dissociate of
approximately 1000.

8. Interpretation of Tandem Mass Spectra of
Peptides
 Known Sequence- Calculate expected fragments and
compare to tandem spectra to see match
 Modified Sequence-Calculate unmodified sequence compare
to tandem spectra to see difference where modification
occurs.
 Unknown Sequence- Check Database to see if it is a match
 Unknown Sequence not in Database- Manual Interpretation
(Practice!Practice!Practice!)

9. Manual Interpretation
 Goal-Assign as many abundant fragments as possible to
a spectra
 Remember Cysteine modifications
 Know type of fragments that are typically observed by
dissociation method.
 Low Energy (b and y, loss of neutrals from these
fragments)
 High Energy (x,y,z, a,b,c, v, d,w)

10. MS/MS: Tandem Mass Spectrometry
Laser, 200
Hz Choice of Different
Collision Gases
(He, Ne, Ar, Kr, Xe)
High CE

11. Conducting
liner
250 L/s
770 L/s
770 L/s
q0 q1 q2
4 anode Razor
detector
Ion Mirror
(reflector)
Sample
Ions
Curtain
Gas
Accelerator
column
Focusing
grid
Q-TOF - Schematics
Effective
Flight
Path = 2.5 m

12. Review: MS/MS Fragment Ion Analysis

13. Peptide fragmentation
-RCH-C-NH-CHR-
O
x y z
cba
Nterm Cterm

14. MS/MS Sequencing of Peptides

15. Average residue masses of amino acids
The side chains that give amino acid its special chemistry are attached to
the alpha carbon.
To complete the structure add an extra proton( 1amu) to the N-terminal
residue and an extra OH (17 amu) to the C- terminal amino acid.

16. MS/MS Spectrum Representation
 For peptide IYEVEGMR
Distance between peaks on m/z axis is used to determine
partial sequence of the peptide

17. Cumulative mass
Peptide sequence
AVAGCAGAR

18. Possible b- and y-ion fragments for the peptide
AVAGCAGAR
The b- ion series complements the y-ion series.
The gap between the b₇ and b₆ ions is 57, which correspond to glycine.
The gap between b₆ and b₅₅ is 71, which corresponds to alanine.
The complement b-ion series(b₈ through b₁) corresponds to the
AVAGCAGA motif.
Thus he y- ion and b-ion series describes the same amino acid sequec in
two different directions.

19. Structures of the b₄ and y₅ ions from cleavage
between the glycine and cysteine residues
B₄ m/z 299.305 Y₅ m/z 476.557

20. Annotated MS-MS spectrum of the [M+2H]²⁺ ion of
AVAGCAGAR showing b- and y- ions

21. Digest protein mixtures
Peptide ions
Experimental MS/MS Spectra
Search databases for peptide
candidates with similar precursor
mass
Theoretical spectra generated
Score candidate peptide
Validate peptide
Highest score: Peptide
identification
Validate protein

22. Charged Parent ion
Fragmentation of the amicle backbone
A, a (no H+ added) X, x (no H+ added)

23. Charged Parent ion
B, b (no H+ added) Y”, y+2 ion or y ion, 2H+ added
Groups formed at cleavage point

24. Charged parent ion
C”, c+2 ion, 2H+ added
Z’, z+1 ion, 1H+ added

25. Charged parent ion
b₂ ion
Immonium ion
Immonium ions
The low mass region of MS/MS spectra often contain
ions that are indicative of the presence of specific amino
acids in the peptides.
These immonium ions arise from at least two internal
bond cleavages.
Labeled with the single letter code for the parent
amino acid.

26. PRTEIN
PRTEYN
PWTEYN
b₁
b₁
b₁
y₁
y₁
y₁
b₂
b₂
b₂
y₃
y₃
y₃y₂
y₂
y₂
b₃
b₃
b₃
b₄
b₄
b₄
y₄
y₄
y₄
b₅
b₅
b₅
y₅
y₅
y₅
100 200 500400300 700600
Theoretical spectral of peptides
PRTEIN and PRTEYN(one mutation) PWTEYN (two mutation)
Representing masses of all the b and y ion in the corresponding peptides.

27. Mechanism of C-terminal cleavage after Asp residue
The carboxylic acid group of Asp
is often involved in
rearrangement reactions.
In the gas phase group can
attack the amide bond giving rise
to a cyclic intermediate that
breaks down to give a b-ion
which often dominates the
MS/MS spectrum.
No C-terminal cleavage is found
for Pro

28. Loss of C-Terminal amino acid
 When the C-terminal amino acid residue is hydrophobic(most
commonly Phe and Tyr and to a lesser extent Leu, Ile, Val) an ion
corresponding to the loss of residue mass is observed in addition
to the y-ion
This is due to intramolecular rearrangement.

29. Low energy production of a-
ions
Charged b ionz
Charged a ionz
Mechanism of a ion formation
Most of the a ion observed in low
energy MS/MS spectra are not
formed by direct cleavage of the
peptide bond as observed in high
energy spectra.
They are formed by loss of CO
giving rise to the formation of a-
and b- ion doublets separated by 28
mass units.

30. Loss of 18,34 and 105 mass units
Loss of water occur very often from serine and threonine
residue.
Cysteine show loss of 34 due to H₂S.
Cysteine is deliberately modified to increase the digestion
efficiency and prevent digetion bond formation.
The most common modifications, with vinylpyridine to form
pyridylethylscysteine or idoacetic(or idoacetamide) to give
carboxymethylcysteine, give rise to losses of 105 and 92
respectively.
Another common loss seen is 48 from metheonine containing
peptides, this is the loss of methylsulphide.

31. Immonium ions
Amino acids Immonium ions Related ions Comments
Ala
Arg
Asp
Asn
Cys
Gly
Gln
Glu
His
Ile/Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Try
Val
44
129
88
87
76
30
101
102
110
86
101
104
120
70
60
74
159
136
72
70,87,100
81
84
61
91
130
107
Marginally useful
m/z 129 weak
other(4:2:1)
Often weak or absent
Often weak
Relatively weak
Not useful
m/z 101 often weak
Very strong
Strong110:81(3:1)
Strong 86:84 (4:1)
Usually weak or absent
Strong 104:61 (3:4)
Strong 120:91 (3:1)
Strong 130:159 (1:2)
Strong 136:107 (1:2)
Fairly strong

32. Relative amino acids mass and their behavior in MS/MS
RESIDUE MASS POSSIBLE EQUIVALENT MASS BEHAVIOUR IN MS/MS
Gly 57 Give weak signal
Ala 71 -
Ser 87 loses -18( water )
Pro 97 strong signal after C-
terminal cleavage
giving internal ions.
Val 99 AcGly (N terminal only)
Thr 101 loses -18( water)
Cys 103 unusual, always
modified ,lose -34
(H2S)
Pyro-Glu 111 N-terminal only
Leu/Ile 113 AcAla (N-terminal only) -
Asn 114 Gly-Gly loses -17(ammonia)

33. RESIDUE MASS POSSIBLE EQUIVALENT MASS BEHAVIOUR IN MS/MS
Asp 115 Gly-Ala Cleave N-terminal to give
strong signals.
Lys 128.09 Gly-Ala
Gln 128.06 Strong lose of -17
Glu 129 AcSer( N- terminal only) -
Met 131 lose of CH₃SH -48
His 137 like Pro but weaker .look
for 110.
Phe 147 Can be Met(Ox)
Arg 156 Gly-Val/AcAsn (N-terminal only)
CmCys 161 -
Tyr 163 No lose of 18
Trp 186 Gly-Glu or Ala-Asp or Ser-Val Like Pro and His but much
weaker internals.

34. Unknown spectrum
Database search
De novo
identification
Compare to model spectra of
complete peptides
Partial sequence

35. Number of peptide candidates
Using enzyme cleavage specificities can
reduce the number of candidates by
approximately one order of magnitude.
Inclusion of additional post-
translational modification can increase
the number of candidates.

36. Manual Interpretation Guide
Accurate mass detection of native parent, methylated and acetylated ions.
Depending on parent charge deconvolute spectrum to all 1+ ions. Remove all
spikes.
Print out spectrum as is , and then with all regions scaled up to the same size.
Label all possible a-b pairs (a=b-28).
Label -18 an -17 losses for water and ammonia respectively.
Examine low mass end.
Look for PHW immonium ions.
If PHW present, look for intense internal cleavage series.
Inspect High mass end for b and y ions and residue mass loss.
Look for ion series for by sequentially subtracting residue masses.
Look for corresponding b/y ion pairs.
For each residue look for diagnostic losses from it and respective immonium
ions.
Be careful about the dipeptide masses that match residue masses.
Assign tentative sequence taking into account acetylation data.
Compare all theoretical masses to data. Are all ions accounted for? If not
repeat the process.