1. Sample Preparation
Enzymatic Digestion
(Trypsin) +
Fractionation

2. LC-MS: 1 MS spectrum / second
Single Stage MS
Mass
Spectrometry
LC-MS: 1 MS spectrum / second

3. Tandem MS Secondary Fragmentation Ionized parent peptide
Tandem mass spectrometry selects one of the intense peaks observed in the single stage mass spectrum and further fragments all peptides with the selected mass to charge ratio. The tandem mass spectrum typically contains mass to charge ratio information about fragments of a a single peptide.
Secondary Fragmentation
Ionized parent peptide

4. Product Ion Scan Mode in a
Triple Quadrupole
Q1 Mass selection
Q2 collision cell
Q3 Full Scan A C B
Ion C
C+Ar Products
Products D
Ions in
Source
Q1 only
transmits
Ion C
Fragment Ion C
Q3 Scans for
products

5. The peptide backbone The peptide backbone breaks to form
fragments with characteristic masses.
H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH
Ri-1 Ri
Ri+1
N-terminus
C-terminus
Tandem MS can be used to determine the amino-acid sequence of a peptide because proteins are made up of amino-acid chains. During secondary fragmentation, the peptide backbone breaks forming fragments with characteristic masses.
AA residuei-1
AA residuei
AA residuei+1

6. Ionization The peptide backbone breaks to form
fragments with characteristic masses. H+
H...-HN-CH-CO-NH-CH-CO-NH-CH-CO-…OH
Ri-1 Ri
Ri+1
N-terminus
C-terminus
The parent peptide, when ionized, has at least one additional proton attached.
AA residuei-1
AA residuei
AA residuei+1
Ionized parent peptide

7. Fragment ion generation
The peptide backbone breaks to form
fragments with characteristic masses. H+
H...-HN-CH-CO NH-CH-CO-NH-CH-CO-…OH
Ri-1 Ri
Ri+1
N-terminus
C-terminus
When the peptide backbone breaks, the ionizing protons are retained on some of the fragments, which can then have their mass to charge ratio measured. Shown here is a suffix fragment, where the ionizing proton is retained on the C-terminus side of the backbone cleavage site. Also possible is a prefix fragment, where the ionizing proton is retained on the N-terminus side.
AA residuei-1
AA residuei
AA residuei+1
Ionized peptide fragment

8. Cleavages Observed in MS/MS of Peptidesbi
yn-i
zn-i
low energy ai
xn-i
vn-i
wn-i
-HN--CH--CO--NH--CH--CO--NH- Ri
CH-R’ R”
high energy ci
di+1

9. Different MS-MS Instruments Yield Different Spectra
A typical QTOF or triple quad MS-MS spectrum of a tryptic peptide contains a continuous series of y-type ions. The b-type ions are usually seen only at lower masses below the precursor m/z value
Ion trap CID data of tryptic peptides is different in that one often finds a continuous series of both b-type and y-type ions throughout the spectrum

10. MS-MS for Protein ID
Proteins are isolated (from gel or HPLC) and subjected to tryptic digestion
Peptides are sent through ionizer and into a collision cell where the doubly charged ions are selected and fragmented through collision induced decay (CID)
The resulting singly charged ions (daughter ions) are analyzed to determine the sequence or to ID the parent peptide

11. Why Trypsin for MS-MS?
CID of peptides less than 2-3 kD is most reliable for MS-MS studies – The frequency of tryptic cleavage guarantees that most peptides will be of this size
Trypsin cleaves on the C-terminal side of arginine and lysine. By putting the basic residues at the C-terminus, peptides fragment in a more predictable manner throughout the length of the peptide

12. Why Double Charges?
Easiest spectra to interpret are those obtained from doubly-charged peptide precursors, where the resulting fragment ions are mostly singly-charged
Doubly-charged precursors also fragment such that most of the peptide bonds break with comparable frequency, such that one is more likely to derive a complete sequence

13. How the peptide sequencing works?
Use Tandem MS: two mass analyzer in series with a collision cell in between
Collision cell: a region where the ions collide with a gas (He, Ne, Ar) resulting in fragmentation of the ion
Fragmentation of the peptides in the collision cell occur in a predictable fashion, mainly at the peptide bonds (also phosphoester bonds)
The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides…
Ser-Glu-Leu-Ile-Arg-Trp
Collision Cell
Ser-Glu-Leu-Ile-Arg
Ser-Glu-Leu-Ile
Ser-Glu-Leu
Etc…

14. MS-MS & Peptide Fragments
When peptides are proteins are admitted to a collision cell the peptide usually fragments at the weakest bond (the peptide bond, but some CH-NH and CH-CO breakage also occurs)
Collision conditions have to be optimized for each peptide
Two main types of daughter ions are produced -- “b” ions and “y” ions

15. MS-MS Peptide Fragmentation
yn-1
yn-2 y1 R1 R2 R3 Rn
H2N-CH-CO-NH-CH-CO-NH-CH-CO…CO-NH-CH-CO2H b1 b2
bn-1
b1 y1 b2 y2 b3 y b4 y4 b5 y5
signal

16. Peptide Fragmentation
E=Glu
G=Gly
S=Ser
F=Phe
N=Asn
P=Pro
V=Val
A=Ala
R=Arg
Peptide Fragmentation
=> E G S F F G E E N P N V A R
175.10
246.14
345.21
459.25
556.30
670.35
799.39
928.43
985.45 = = =

17. MS-MS Peptide Fragmentation
Ala-Gly-His-Leu-….Phe-Glu-Cys-Tyr
b1 y1 b2 y2 b3 y b4 y4 b5 y5
signal

18. Peptide sequencing by mass spectrometry
N-term. A B C D E
C-term.
Peptide molecules are fragmented by collisionally activated dissociation (CAD)
collisions with neutral background gas molecules (nitrogen, argon, etc)
typically dissociate by cleavage of -CO-NH- bond A B A B C D A A B C A B C D E
m/z
N-terminal product ions

19. Peptide Sequencing

20. Protocols for MS-MS Sequencing
Usually can’t tell a “b” ion from a “y” ion
Assume the lowest mass visible in the spectrum is a lysine or arginine (this is the y1 ion) this is because trypsin cuts after a lysine or arginine
This y1 mass should be for lysine or for arginine {The y1 ion is calculated by adding u (three hydrogens and one oxygen) to the residue masses of lysine and arginine}

21. MS-MS Sequencing
Use the remaining “unassigned” peaks to see if you can construct a “b” ion series
The highest mass peak corresponds to the parent ion or parent minus 147 (K) or 175 (R)
The “b” ions give the “normal” sequence
Both forward (b ion) and backward (y ion) sequences should be consistent
Use the resulting sequence tag to search the databases using BLAST (remember to use a high Expect value ~ 100) to see if the sequence matches something

22. Peak Assignment 88 145 292 405 534 663 778 907 1020 1166 b ions S G FL E E D E L K
1166
1080
1022
875
762
633
504
389
260
147
y ions y6
100
Peak assignment implies
Sequence (Residue tag)
Reconstruction! y7
% Intensity
[M+2H]2+
An assignment of the peptide fragments to the spectral peaks. You can see there is an excellent correspondence between the fragments that are expected and the observed spectral peaks. Note too the presence of the doubly charged, unfragmented parent ion. y5 b3 b4 y2 y3 y4 b5 y8 b6 b8 b9 b7 y9
250
500
750
1000
m/z

24. Peptide Sequencing by Mass Spectrometry
LVDKVIGITNEEAISTAR
Cysteine Synthase A
242
259
b3-17
y12
1261.4
100
y10
1091.5
y13
1374.5
Rel. Abund. b6
668.4 b8 b5 y9
838.5
y14
555.4
990.5
y11 y5 y4 b7
1474.4 b4 b9 b3 y6 y8 y7
b10
b11
b14
b12
b13
b15
b16
b17
400
600
800
1000
1200
1400
1600
1800
m/z

25. “+2” spectra

26. “bad” spectra

27. Modifications
Some residues may be modified during the sample preparation procedure
This introduces discrepancies in the expected and observed masses
For example, Met residues are often oxidized

28. Mass of PTMs Mass Change Modification +14.0 Methylation
Methylation
Hydroxylation
Formylation
Nitrosylation
Acetylation
Sulfation
Phosphorylation
Mono-glycosylation
Farnesylation
Myristoylation

29. MS/MS of Peptides Containing Phosphotyrosine
200
400
600
800
1000
1200
1400
1600
m/z
100 % K R Y
415.26
201.14
951.50
708.46
545.39 D T E L
G, G Yp y1 y3 y4 y5 y6 y7 y8 y9
y10
y11
y12 MH
163
243
Relative Intensity