Analytical strategy

2D gel In-gel digestion and MALDI-TOF Search protein database

peptide
fingerprint

Tandem MS (MS/MS)
Search DNA database
peptide sequencing Sequence 1
MS-MS
Sequence 2…...
BLAST
Degenerate
DNA sequence

Design oligonucleotide probe Search EST or genome

for gene cloning from cDNA database for obtaining novel
library genes

De novo sequencing by tandem mass spectrometry (MS-MS)

Some important databases for protein identification
Program Name Internet address 1. Various types of tandem MS
Triple quadrupole, Q-TOF, TOF-TOF, Ion-trap
programs that use amino acid sequences for the search query
2. Collision-induced dissociation
FASTA fasta.bioch.virginia.edu/fasta (CID)
BLAST www.ncbi.nlm.nih.gov/blast.cgi
MS-Edman prospector.ucsf.edu/mshome3.2.htm Ar
programs that use peptide molecular weights for the search query
MS-Fit prospector.ucsf.edu/mshome3.2.htm
MOWSE srs.hgmp.mrc.ac.uk/cgi-bin/mowse
Peptidesearch www.mann.embl-heidelberg.de/Services/PeptideSearch/
PeptideSearchIntro.html
MASCOT
programs that use uninterpreted product ion spectra for the search query
SEQUEST thompson.mbt.washington.edu/sequest/
MS-Tag prospector.ucsf.edu/mshome3.2.htm
Peptidesearch www.mann.embl-heidelberg.de/Services/PeptideSearch/
PeptideSearchIntro.html

MALDI MS/MS Analysis

Technical Comments
LID CID
zLID:
z Laser Induced Decomposition: the laser “puts”
so much energy into a molecule that the molecule
“fragments”.

zCID:
z Collision Induced Decomposition: molecules fly
through a collision cell and fragment as a result of
collisions with collision gases.
z LID and CID are used in the TOF-TOF as two
alternative and complementary dissociation
methods.

1
 Peptide Fragmentation and Ionization

TOF-TOF

Ar (CID)

Tandem Mass Spectrum

MS/MS Analysis
z Also known as tandem MS
z Individual peptides from the enzymatic
digests are fragmented further
z From this “ladder” sequences may be
reconstructed

MS/MS Analysis MS/MS Analysis

z Much more discriminating search than
z Much more discriminating search than
simple peptide mass fingerprinting simple peptide mass fingerprinting

intensity Peptide
Mass spectrum database Mass spectrum spectrum from
experiment
Protein A
m/z

Protein B
Theoretical
intensit spectrum of a
y peptide
Protein C

m/z

2
 MS/MS Analysis ESI MS/MS Analysis
z Much more discriminating search than
z Carried out on nanospray/electrospray mass
simple peptide mass fingerprinting
spectrometers
intensity Peptide z Rather than spotted on a target plate, the
Mass spectrum spectrum from
experiment
sample is introduced through an inlet from a
capillary
m/z

1.Tandem in space
2.Tandem in time

Tandem in space
Micromass QTOF

Tandem in time
Finnigan Ion Trap

3
 Daughter Ions Peptide fragmentation
z Unlike the MALDI, ions produced by
electrospray/nanospray machines may carry
multiple charges
z Various types of ions are produced,
categorized by their charge and their
direction in the peptide sequence
z Fortunately the peptides fragment at the
peptide bonds

B and Y Fragment Ions

Y-ions from C to N terminus
Y ion Y ion Y ion
3 2 1
R1 O R2 O R3 O R4 O

NH 2 C
H
C N
H
C
H
C N
H
C
H
C N
H
C
H
C OH MS/MS Search programs
B1 ion B ion B ion
2 3

B-ions from N to C terminus

MS/MS Search programs MS/MS Search programs

z In a similar fashion to peptide mass
MASCOT fingerprinting, the predicted fragment ion
http://www.matrixscience.com/ mass from each peptide of a database
MS-TAG sequence are calculated
http://prospector.ucsf.edu/ Mass
spectrum
SEQUEST
http://fields.scripps.edu/sequest/
Database

4
 MS/MS Search programs Problems with MS/MS data
Residue modifications are far more difficult
z The calculated and observed ion masses are to handle, the number of mass permutations
compared and given a score being very large
z Individual peptide scores are combined to
give a protein score
K
K E
Mass
spectrum E T
T oxidation
M
M
P P
Database I I
M M
A A

Theoretical fragmentation of peptide

MS/MS Search programs
Mass
spectrum

Database

Protein database
EST database
Genome database

Problems with MS/MS data

Peptides that span exon splices will be missed
when matching uninterpreted MS-
MS-MS data to
genomic DNA
MASCOT Searches with
MS/MS Data

5
Finnigan Ion Trap

Dp1806.asc

Mass
spectrum

6
Sequest Searches with
MS/MS Data

7
Database Manager Indexed Databases
SEQUEST’s unique Database
Indexed Databases Lead to Rapid and
Manager allows you to create indexed
Efficient
databases, subset databases, or new databases
from a protein/RNA/DNA sequence. Protein Identification

LC-LC-MS-MS for Complex Mixtures

peptides
Results in thousands of mass
from many spectra
proteins A computational challenge!

8
MS/MS Method Using ESI

A Peptide fragmentation files Multiple Peptide fragmentation files

9
SEQUEST summary output example SEQUEST summary output example
Protein summary
peptides XCorr proteins

De-Novo Sequencing
z If the protein is still not identified, the
sequence of a peptide has to be
reconstructed from the MS/MS data
De-Novo Sequencing z Very time consuming and demands a great
deal of skill, noisy data is very problematic
z Sequencing is carried out by finding mass
differences between peaks that correspond
to amino acid masses

Tags
z Easy to find initial masses in “ladder”
z Tags modify the fragmentation of the
peptide
z Reduce isobaric problems
z Neutralise the adverse effects of certain
residues on peptide fragmentation

10
 Databases Searched with
MS/MS Data
z Non-identical protein databases are the ideal
z EST databases translated in 6 frames are
very useful as individual peptides may be
identified
z Translated nucleic acid databases
z Non-redundant databases create problems

Example Tags Example Tags

C-Termina1 180 Labelling
Esterification

Example Tags Example Tags

O O H

Pep NH 2 + N N R
R O Pep
O
O

R=
N N
O
N N
CH 2 CH 2 CH 2

Pyridine Imidazol Morpholine

11
 A b-type ion graph for peptide sequencing
Ala-Ala-Lys (mw. 288) 288 + 2 proton

Amino Acid Mass Table Basic Computing Scheme

A 71.08 M 131.19
peptide mass W tandem mass spectrum S
C 103.14 N 114.1
D 115.09 P 97.12
E 129.12 Q 128.13 NC spectrum graph
F 147.18 R 156.19
G 57.05 S 87.08 Find feasible paths to order the masses
in S to identify all the b-ions and y-ions
H 137.14 T 101.11
consistent with S.
I 113.16 V 99.13
K 128.17 W 186.21
L 113.16 Y 163.18 Convert feasible paths into legal peptide sequences

NC-
NC-Spectrum Graph: Nodes (1) NC-
NC-Spectrum Graph: Nodes (2)

Assumption 1: Assumption 2:
If Ion 1 is an y-ion If Ion 1 is a b-ion
Ion # 1 (274.11) N1: a b-ion node
C1: a b-ion node

N0 C0 N0 C1 N1 C0

0 429.22 0 174.11 273.11 429.22

mass of this peptide mass of this peptide

mass( ) + mass( ) = mass(P) + 18

12
 NC-
NC-Spectrum Graph: Nodes (3) NC-
NC-Spectrum Graph: Edges (1)

Ion # 2 (88.10) Mass(S) = 87.08.

S
N0 N2 C1 N1 C2 C0 N0 N2 C1 N1 C2 C0
0 87.10 174.11 273.11 360.12 429.22 0 87.10 174.11 273.11 360.12 429.22

mass( ) + mass( ) = mass(P) + 18

NC-
NC-Spectrum Graph: Edges (2) NC-
NC-Spectrum Graph: Edges (3)

Mass(W) = 186.21 Mass(W) = 186.21

Mass(S) = 87.08. Mass(S) = 87.08.
W W
S S
N0 N2 C1 N1 C2 C0 N0 N2 C1 N1 C2 C0
0 87.10 174.11 273.11 360.12 429.22 0 87.10 174.11 273.11 360.12 429.22

S+W

Mass(S+W) = 273.29

NC-
NC-Spectrum Graph: Edges (4) NC-
NC-Spectrum Graph

Mass(W) = 186.21 Mass(R) = 156.19

Mass(S) = 87.08.
W R
S
N0 N2 C1 N1 C2 C0 N0 N2 C1 N1 C2 C0
0 87.10 174.11 273.11 360.12 429.22 0 87.10 174.11 273.11 360.12 429.22

S+W

Mass(S+W) = 273.29

13
 NC-
NC-Spectrum Graph: Paths = Sequences NC-
NC-Spectrum Graph: A Feasible Path (1)

b-ions

W R a feasible path W R
S S
N0 N2 C1 N1 C2 C0 N0 N2 C1 N1 C2 C0
0 87.10 174.11 273.11 360.12 429.22 0 87.10 174.11 273.11 360.12 429.22

Definition: A feasible path is a path from N0 to C0 that goes

b-ions through exactly one node for each pair (either Nj or Cj).

NC-
NC-Spectrum Graph: A Feasible Path (2) NC-
NC-Spectrum Graph: Not A Feasible Path (1)

not a feasible path:

b-ions y-ions
(1) miss ion #2
a feasible path
S S
N0 N2 C1 N1 C2 C0 N0 N2 C1 N1 C2 C0
0 87.10 174.11 273.11 360.12 429.22 0 87.10 174.11 273.11 360.12 429.22

GVV
Definition: A feasible path is a path from N0 to C0 that goes Definition: A feasible path is a path from N0 to C0 that goes
through exactly one node for each pair (either Nj or Cj). through exactly one node for each pair (either Nj or Cj).

NC-
NC-Spectrum Graph: Not A Feasible Path (2) NC-
NC-Spectrum Graph: Not A Feasible Path (3)

not a feasible path: not a feasible path:

(2) repeat ion #1 (1) miss ion #2
(2) repeat ion #1

N0 N2 C1 N1 C2 C0 N0 N2 C1 N1 C2 C0
0 87.10 174.11 273.11 360.12 429.22 0 87.10 174.11 273.11 360.12 429.22

Definition: A feasible path is a path from N0 to C0 that goes Definition: A feasible path is a path from N0 to C0 that goes
through exactly one node for each pair (either Nj or Cj). through exactly one node for each pair (either Nj or Cj).

14
15