Download Seminar on Protein Sequencing

Automated Techniques for
High Throughput
Protein Identification & Peptide
Sequencing
Lori L. Smith
October 27, 1999
2D Polyacrylamide Gel Electrophoresis
Normal
Heart Tissue:
Distressed
Heart Tissue:
Proteins Present in Normal Tissue
OR
A Novel Protein?
Human Genome Project
October 1, 1988
NIH
DOE
Foundation for
future research
in human
genetics and
biology
Improve ability
to assess
effects of
radiation and
energy-related
chemicals on
human health
Genetics
Molecular
Biology
Medicine
Computer
Science
Engineering
Major Goals
Mapping Genomes and DNA Sequencing
E. Coli / Yeast
Roundworm
Fruit Fly
Mouse
Human
100,000 genes!
Technology Development
Ethical, Legal and Social Implications (ELSI)
Projected Completion Date: 2003
DNA-containing
Chromosomes
mRNA
Ribosome
Protein
DNA is not the true “bottom line”.
DNA
Gene 1
Gene 2
Gene 3
Protein 1
Protein 2
Protein 3
Genome
Proteome
Genomics vs. Proteomics
Genomics
Proteomics
Relationships between Gene
Activity and Particular Diseases
Why Study Proteins?
Poor Correlation between Gene
Activity and Protein Abundance
Protein Modifications aren’t
Coded by Genes
Protein Level Events
(i.e. ACE inhibitors for lowering blood pressure)
Digest
Protein
Peptides
Peptide Structure
N-Terminus
C-Terminus
R1 O
R2 O
R3 O
H2N-CH- C-HN-CH-C-HN-CH-C-OH
“Peptide Bond”
R O
H2N-CH-C-OH
20 Natural Amino Acids
R group
Amino Acid
Arginine (R)
H2N
C NH (CH2)3
HN
Aspartic Acid (D)
Serine (S)
Alanine (A)
HO
C
O
HO
CH2
CH2
CH3
Protein Digestion
...MRLLPLLALLA...
CNBr
(Met)
Trypsin
(Lys or Arg)
Pepsin
(Phe, Leu,
Tyr, Trp)
...M
RLLPLLALLA...
...MR
LLPLLALLA...
...MRL
...MRLL
...MRLLPL
Etc.
Edman Degradation Reaction Cycle
Goal:
Identification of Isolated Protein by
Sequence Determination of Peptides
O
PITC
+
N C S
H2N
CH
R1
C
Peptide
NH
Alkaline pH
S
Excess PITC
Extracted
O
N C HN
Peptide
C
CH
H
NH
R1
Strong Anhydrous
Acid
PTH-AA
S
C
N
NH
C CH
O
R1
ATZ +
Derivative
H2N Peptide
Advantages
Most Reliable Sequencing Technique
Limitations
Need Pure Samples of Peptides (> 2 pmole)
Requires 40-60 min / Amino Acid
Can’t Analyze N-Terminally Modified Peptides
O
H2N
CH
R1
C
O
Peptide
NH
vs.
H3C
C
O
HN
CH
R1
C
Peptide
NH
Low Concentrations: His, Arg, Cys, and Trp
2000
Mass Spectrometry
Protein Mixtures
Minimal
Separation
Isolate
Protein
Digest Protein(s)
Peptide Mass
Mapping
Tandem MS
Peptide Mass Mapping
Goal:
Rapid Identification of Isolated Proteins
PeptideSearch - Mathias Mann, et al. Biological Mass Spectrometry, (1993) Vol. 22, pg. 338-45
MOWSE - Pappin et al., Current Biology, (1993), Vol. 3, No. 6, pg. 327
2D PAGE
Prep.
Sample
Peptides
Cut
Digest
Isolate
...MRLLPLLALLA...
Sequences from
Database
Peptides
MALDI-MS
“Digest”
3
1
2
5
List of Theoretical
Peptide Masses
Peptide Masses
Protein Match
Advantages
Direct Mass Measurement
Automated; Very Fast Analysis
(< 1 Minute)
Method Improvement with
Database Increase
Limitations
Proteins Best Matched from Organisms
with Completed Genomes
No Protein Mixtures w/o Prior Separation
Protein Mixtures
Isolate
Protein
Minimal
Separation
Digest Protein(s)
Peptide Mass
Tandem
Mapping
Isolate Peptides
MS
Sequence Tags
De Novo Sequencing
SEQUEST
Tandem Mass Spectrometry
Ionization
Analysis
MS:
Ionization
Fragmentation Analysis
MS/MS:
Activation
Collision Induced Dissociation (CID)
Ar
Ar
Peptide
Ion
Energy
Transfer
Ar
Ar
F
Ar
F
Ar
Surface Induced Dissociation (SID)
Peptide
Ion
F
F
Chemically
Modified
Surface
Fragmentation Ion Nomenclature
a1
b1
R1 O
R2 O
R3 O
H2N-CH- C-HN-CH-C-HN-CH-C-OH
z2
x2
y2
Immonium Ions:
+
H2N=CHR
Peptide Sequencing by MS/MS
R1 O
R3 O
R2 O
R4 O
H2N-CH-C-HN-CH-C-HN-CH-C- HN-CH-C-OH
H+
+
H
b3
b2
b1
y3
y2
y1
Peptide Sequence Tagging
Goal:
Rapid Identification of Proteins by a
Portion of the AA Sequence
PeptideSearch - Mathias Mann, et al. Biological Mass Spectrometry, (1993) Vol. 22, pg. 338-45
m1
Sequence Tag
M
I/L
N
m3
m1 = 920.5
Tag = MIN
m3 = 515.2
Computer Search String:
(920.5)MIN(1287.7)
Peptide MW = 1802.9
Peptide
m1
Sequence Tag
M
I/L
m3
N
...
...
Sequence from Database
Advantages
Peptide MW + 3 AAs
1 out of 100,000
Can Be Used w/ Any Sequencing
Technique
Search Time is Only 5-20 sec
Limitations
Can Only Match to Proteins in
Database
Human Intervention Required
Peptide Sequencing by MS/MS
R1 O
R3 O
R2 O
R4 O
H2N-CH-C-HN-CH-C-HN-CH-C- HN-CH-C-OH
H+
+
H
b3
b2
b1
y3
y2
y1
b3
a1
F
L
GF
a4
b2
y3
MH+
b4
m/z
Data Interpretation
Rate-Determining Step
YGGFL
Predominant Reactions
k(,)
Activation Process and
Energy Deposited
Instrument (Time Frame)
Automated Spectral Interpretation
Peptide
Sequence
SEQUEST
Goal:
Rapid Identification of Proteins by
MS/MS Peptide Sequencing w/
Automated Spectral Interpretation
SEQUEST - Eng JK, McCormack AL, Yates JR III. Journal for the American Society for Mass
Spectrometry, (1994), Vol. 5, pg. 976-989.
Tandem MS Data Reduction
100%
2
3
Immonium
Ion(s)
1
Nominal m/z
200 Most
Abundant
Search Database
Experimental Tandem
Mass Spectrum
m/z
...Gly Ser Asp...
...Leu Ile Phe...
...Ala Arg Gln...
Computer
Comparison
...Lys Trp His...
Sequence
Database
Predicted MS/MS
Advantages
Can Analyze Protein Mixtures
w/o Prior Separation
Ease of Automation
Very Fast; “Real Time” Analysis
Program can be Redesigned to
Include Covalent Modifications and
Fragmentation Mechanisms
Limitations
Very Few Fragmentation “Rules”
Conclusions
Peptide Mass Mapping:
One Separation Step (Isolate Protein)
Create Spectrum of Peptide Masses
Search Database for Corresponding Protein
Peptide Sequence Tagging:
No Separation
Manual Interpretation of Sequence Tag
Search Database for Protein
(Peptide MW, Sequence Tag, m1/m3 Criteria)
De Novo vs. SEQUEST
Experimental
Spectrum
De Novo Manipulated
Not
SEQUEST
Manipulated
Peptide
Sequence
Protein
Identification
Statistically
Generated
Use 2nd Algorithm
to Search Database
w/ Sequences
Generated by
Comparing
Predicted Spectra
to Experimental
Spectra
Inherent in
Peptide
Sequence
Determination
2009
Improvement / Development of
Separations Techniques
Robotics Incorporation for Sample
Preparation
Adding Fragmentation Mechanisms to
Spectral Interpretation Programs
Fully Automated Protein Sequencing
Acknowledgements
Seminar Committee
Dr. Wysocki
Dr. Saavedra
Dr. Baldwin
Wysocki Group
Vince Angelico
Linda Breci
George Tsaprailis
Darrin Smith
Emily McAlister
Joey Robertson
Domenic Tiani
Baldwin Group