Recap of Tuesday
• mass spectrometry
– make ions (maldi or ESI)
– analyze ions (1 example is ion trap)
• proteins
– analyze them by mass spectrometry
– look up information about them
• complex protein mixtures
– separate by gel electrophoresis methods
– analyze very complex mixtures (MUDPIT)
• single stage mass spectrometry (MS)
– measure all peptides in one spectrum
– produces low confidence results
Today – protein identification
by tandem mass spectrometry
• tandem mass spectrometry (MS/MS)
– measure peptides as they elute from an HPLC
– ESI-Ion Trap
– produces high confidence results
• homology searching (BLAST)
1
MS/MS Method using ESI-Ion Trap
HPLC – MS/MS Ion Trap
UV Trace
Chromatogram
Relative Intensity
MS at 25.2 Min
100
100
80
80
60
60
40
40
20
20
0
200
400
600
800
1000
1200
m/z
1400
1600
1800
2000
0
200
MS-MS of
m/z 1574.2
400
600
800
1000
1200
1400
1600
1800
2000
m/z
2
895.25
Result is many MS/MS spectra
How do we determine the aa sequence?
Peptides fragment in a predictable way
H
O
O
NH
H2N
NH
O
+ N-terminal = a/b ions + neutral
or
+ C-terminal = neutral + y ion
OH
N
NH
O
O
b2
a2
R1
Doubly charged parent?
possible b/y ion pair
b3
a3
O
R3
O
NH
NH
H2N
OH
N
O
R5
R2
NH
O
y3
R4
O
y2
3
Peptides fragment in a predictable way
H
O
O
NH
H2N
NH
+ N-terminal = a/b ions + neutral
or
+ C-terminal = neutral + y ion
OH
N
NH
O
O
O
b2
a2
R1
Doubly charged parent?
possible b/y ion pair
b3
a3
O
R3
O
NH
NH
H2N
OH
N
O
R5
NH
R2
R4
O
y3
O
y2
Masses of amino acids between peptide bonds
amino acid
71 u.
115 u.
Ala
O
C
Asp
O
H
N
CH
CH3
C
O
H
N
CH
C
CH2
C
OH
O
H
N
mass
Alanine
ALA
A
71.09
Arginine
ARG
R
156.19
Aspartic Acid
ASP
D
115.1
Asparagine
ASN
N
114.09
Cysteine
CYS
C
103.15
Glutamic Acid
GLU
E
129.12
Glutamine
GLN
Q
128.14
Glycine
GLY
G
57.05
Histidine
HIS
H
137.14
Isoleucine
ILE
I
113.16
Leucine
LEU
L
113.16
Lysine
LYS
K
128.17
Methionine
MET
M
131.19
Phenylalanine
PHE
F
147.18
Proline
PRO
P
97.12
Serine
SER
S
87.08
Threonine
THR
T
101.11
Tryptophan
TRP
W
186.12
Tyrosine
TYR
Y
163.18
Valine
VAL
V
99.14
4
An MS spectrum
Each peak is from a peptide
[M+ 2H]2+
MH+
895.25
5
Data cannot be manually analyzed,
instead, computer programs do it
?
MS/MS
protein
identify
peptides
rank
120000
compare
100000
Relative Intensity
80000
Relative Intens ity
MS Peptide MW
Found in Selected
Databases
NDALYFPT...
SWDLTAL...
PTDLDVSY...
60000
40000
200
400
600
800
1000
1200
1400
1600
m/z
20000
0
theoretical spectra
200
400
600
800
1000
1200
1400
1600
m/z
More peptides identified
increases confidence in ID
VFGTDMDNSR
IFDDSDQTK
LVNLGK
QAEDVNLLDQMSK
If all of these peptides belonged to an
unknown protein, MS/MS could
potentially reveal protein identity
6
gi|126509|sp|P07740|LUXA_VIBHA Alkanal monooxygenase alpha chain
(Bacterial luciferase alpha chain)
MKFGNFLLTYQPPELSQTEVMKRLVNLGKASEGCGFDTVWLLEHHFTEF
GLLGNPYVAAAHLLGATETLNVGTAAIVLPTAHPVRQAEDVNLLDQMSKG
RFRFGICRGLYDKDFRVFGTDMDNSRALMDCWYDLMKEGFNEGYIAADN
EHIKFPKIQLNPSAYTQGGAPVYVVAESASTTEWAAERGLPMILSWIINTHE
KKAQLDLYNEVATEHGYDVTKIDHCLSYITSVDHDSNRAKDICRNFLGHWY
DSYVNATKIFDDSDQTKGYDFNKGQWRDFVLKGHKDTNRRIDYSYEINPV
GTPEECIAIIQQDIDATGIDNICCGFEANGSEEEIIASMKLFQSDVMPYLKEK
Q
Computer Exercise #5
Identify this same protein using MS-Tag
MS-MS of 571.4
Scan #316-318
Enter m/z values
found in excel
7
Computer Exercise #7
• Identify unknowns using the method of #5
Open source software for high-throughput
proteomics: X Tandem
• Current trends to free software
• The Global Proteome Machine http://www.thegpm.org/
– X Tandem
– Sequenced peptide libraries
– Software available to programmers
http://www.u.arizona.edu/~breci/RCE_data.html
8