Transcript Mass Spectrometry of Peptides - Central Web Server 2

Peptide Sequencing by
Mass Spectrometry
Alex Ramos
5 April 2005
Edman degradation
Phenyl isothiocyanate
N
C
S
+
H2N
H
O
C
C
O
Asp
Phe
Phe
Arg
C
O
CH3
Labeling
H
S
H
H
O
N
C
N
C
C
O
Asp
Phe
Phe
Arg
C
CH3
O-
Release
S
N
O
CH3
N
O
H
PTH-alanine
+
H2N
Asp
Phe
Phe
Arg
C
O-
Peptide shorthened by one residue
-
Edman Degradation v. MS/MS
Why study proteins?



machines that make cells function
RNA levels do not always accurately predict
protein levels
targets of drugs
Peptide Analysis


Edman Degradation
MS




More sensitive
Can fragment peptides faster
Does not require proteins or peptides to be purified to
homogeneity
Has no problem identifying blocked or modified proteins
Introduction



MS/MS plays important role in protein identification (fast
and sensitive)
Derivation of peptide sequence an important task in
proteomics
Derivation without help from a protein database (“de novo
sequencing”), especially important in identification of
unknown protein
Basic lab experimental steps
1. Proteins digested w/ an enzyme to produce peptides
2. Peptides charged (ionized) and separated according
to their different m/z ratios
3. Each peptide fragmented into ions and m/z values of
fragment ions are measured

Steps 2 and 3 performed within a tandem mass
spectrometer.
Mass spectrum



Proteins consist of 20 different types of a. a. with
different masses (except for one pair Leu and Ile)
Different peptides produce different spectra
Use the spectrum of a peptide to determine its
sequence
Objectives


Describe the steps of a typical peptide analysis
by MS (proteomic experiment)
Explain peptide ionization, fragmentation,
identification
Why are peptides, and not proteins,
sequenced?



Solubility under the same conditions
Sensitivity of MS much higher for peptides
MS efficiency
MS Peptide Experiment
Choice of Enzyme
Cleaving
agent/Proteases
Specificity
A. HIGHLY SPECIFIC
Trypsin
Endoproteinase Glu-C
Endoproteinase Lys-C
Endoproteinase Arg-C
Endoproteinase Asp-N
B. NONSPECIFIC
Chymotrypsin
Thermolysin
Arg-X, Lys-X
Glu-X
Lys-X
Arg-X
X-Asp
Phe-X, Tyr-X, Trp-X, Leu-X
X-Phe, X-Leu, X-Ile, X-Met, X-Val, X-Ala
3 nS LASER PULSE
MALDI
+++
+
++
++
+
++
+
+
++
++
++
+
+
++
Sample (solid) on target at
high voltage/ high vacuum
+
+
++
+
+
+
+
++
+
++
++
+
+
+
++
+
++
+
+
++
++
++
+
+++ +
++
++
+++
++
+
+
+
+++
++ +
++
+++
+
++
+++++
+
++ +
+++
+++
+
+
TOF analyzer
High vacuum
MALDI is a solid-state technique that gives ions in pulses,
best suited to time-of-flight MS.
ESI
Liquid flow
Q or Ion Trap
analyzer
Atmosphere Low vac. High vac.
ESI is a solution technique that gives a continuous stream of ions,
best for quadrupoles, ion traps, etc.
….MALDI or Electrospray ?
MALDI is limited to solid state, ESI to liquid
ESI is better for the analysis of complex mixture as it is
directly interfaced to a separation techniques (i.e. HPLC or
CE)
MALDI is more “flexible” (MW from 200 to 400,000 Da)
Protein Identification Strategy
*
I
12
Peptides
Protein
mixture
14
Time (min)
16
1D, 2D, 3D peptide separation
10-Mar-200514:28:10
CAL050310A 71 (1.353) Cm (1:96)
II
*
TOF MSMS 785.60ES+
2.94e3
684.17
100
333.15
813.16
480.16
785.62
%
1285.14
1056.17
942.16
685.18
814.17
187.07
246.13
175.12
1171.14
1057.18
497.09
740.09
627.17
612.08
286.11 382.11
943.17
200 400 600 80010001200
Q1
Q2
Collision Cell
Q3
10-Mar-200514:28:10
CAL050310A 71 (1.353) Cm (1:96)
III
Correlative
sequence database
searching
785.62
%
814.17
1171.14
1057.18
497.09
627.17
612.08
286.11 382.11
480.08
943.17
740.09
924.16
498.09
1285.14
1056.17
942.16
685.18
169.06
1039.13 1058.17
1286.14
1172.15
1173.16
1287.13
1296.10
1038.17
0
200
300
400
500
500
600
700
800
900
1000
1100
1200
1300
1400
1500
700
800
900
1000
1296.10
1100
TOF MSMS 785.60ES+
2.94e3
684.17
813.16
480.16
814.17
1171.14
1057.18
497.09
627.17
612.08
286.11 382.11
480.08
169.06
943.17
740.09
924.16
498.09
1285.14
1056.17
942.16
685.18
187.07
246.13
175.12
1039.13 1058.17
1286.14
1172.15
1287.13
1173.16
1296.10
1038.17
0
m/z
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
200 400 600 800 10001200
m/z
Theoretical
785.62
%
1600
200 400 600 800 10001200
m/z
Protein identification
1287.13
m/z
600
CAL050310A 71 (1.353) Cm (1:96)
100
m/z
100
400
813.16
480.16
187.07
246.13
175.12
300
1173.16
1200
1300
1400
1500
1600
Tandem mass spectrum
333.15
333.15
200
1039.13 1058.17
1038.17
10-Mar-200514:28:10
100
TOF MSMS 785.60ES+
2.94e3
684.17
100
100
924.16
498.09
480.08
m/z
169.06
0
1286.14
1172.15
Acquired
Breaking Protein into Peptides and
Peptides into Fragment Ions
Proteases, e.g. trypsin, break protein into
peptides
 MS/MS breaks the peptides down into fragment
ions and measures the mass of each piece


MS measure m/z ratio of an ion
Peptide fragmentation
Amino acids
differ in their
side chains
Weakest bonds
Predominant
fragmentation
Tendency of peptides to fragment at Asp (D)
C-terminal side of Asp
Mass Spectrometry in Proteomics
Ruedi Aebersold* and David R. Goodlett
269 Chem. Rev. 2001, 101, 269-295
Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass
Spectrometry, Molecular & Cellular Proteomics, 2003, 2.11, 1234, Thomas S. Nuhse, Allan Stensballe, Ole N. Jensen, and Scott C.
Peck
What you need for peptide mass mapping

Peptide mass spectrum

Protein Database


GenBank, Swiss-Prot, dbEST, etc.
Search engines

MasCot, Prospector, Sequest, etc.
Database search for protein identification
Protein Identification by MS
Library
Spot removed
from gel
Artificial
spectra built
Fragmented
using trypsin
Spectrum of
fragments
generated
MATCH
Artificially
trypsinated
Database of
sequences
(i.e. SwissProt)
Conclusions


MS of peptides enables high throughput
identification and characterization of proteins in
biological systems
“de novo sequencing” can be used to identify
unknown proteins not found in protein databases
References
H. Steen and M. Mann. “The ABC’s (and XYZ’s) of Peptide
Sequencing” Molecular Cell Biology, Nature Reviews.
2004, 5, 699.
T. S. Nuhse, A. Stensballe, O. Jensen, and S. Peck. “Largescale Analysis of in Vivo Phosphorylated Membrane
Proteins by Immobilized Metal Ion Affinity Chromatography
and Mass Spectrometry” Molecular & Cellular Proteomics,
2003, 2.11, 1234.
R. Aebersold and D. Goodlett. “Mass Spectrometry in
Proteomics” Chem. Rev., 2001, 101, 269.