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Protein and Peptide Sequencing by FTMS
Susan Martin
Protein and Peptide Sequencing by FT-ICR MS
Susan E. MartinUniversity of Virginia, Charlottesville, VA 22901
The University of the Sciences in PhiladelphiaDepartment of Chemistry & Biochemistry
Philadelphia, PA 19104
Office phone 215-596-8551 email: s.martin @ usip.edu
Peptide Fragmentation
O
NH
R2O
H3N
R 1 O
NH
R 4O
NH
R 3
OH
b
y
+
Fragmentation of Tryptic Peptide
m/z
147K
1166L
260
1020E
389
907D
504
778E
633
663E
762
534L
875
405F
1022
292G
1080
145S
1166
88
y ions
b ions
% R
elat
ive
Ab
un
dan
ce
100
0250 500 750 1000
y2 y3 y4
y5
y6
y7
b3b4 b5 b8 b9
[M+2H]2+
b6 b7 y9
y8
Protein Sequencing by Mass Spectrometry
PROTEIN SAMPLE
DIGESTION
HPLC SEPARATION
DISSOCIATION
SEQUENCE
Advantages of FT-ICR MS for Protein Analysis
• Ultra high resolution
• Accurate mass measurement
• High sensitivity
800 900 1000 1100 1200 1300m/z
+10
+11
+12+13+14
+15
994.5 995.0
Exact Mass Determination for APV-1
11924.214 (exp.)11924.203 (cal’d)
% A
bund
ance
100
0
800 900 1000 1100 1200 1300m/z
+10
+11
+12+13+14
+15
994.5 995.0
Exact Mass Determination for APV-1
11924.214 (exp.)11924.203 (cal’d)
% A
bund
ance
100
0
800 900 1000 1100 1200 1300800 900 1000 1100 1200 1300m/z
+10
+11
+12+13+14
+15
994.5 995.0
Exact Mass Determination for APV-1
11924.214 (exp.)11924.203 (cal’d)
% A
bund
ance
100
0
Advantages of FT-ICR MS for Protein Analysis
• Ultra high resolution• Accurate mass measurement
• High sensitivity
• MSn capability1. Collision activated dissociation
2. IR photodissociation
Collision Activated Dissociation
1. Precursor isolation (SWIFT)
2. Precursor excitation (SORI)
3. Collision with Argon at 1x10-6 torr4. Pump out delay (30 s.)
5. Excite and detect
IR Photodissociation
1. Precursor isolation (SWIFT)
2. Single laser pulse (40W cw CO2 laser)
3. Excite and detect
CAD vs. IRMPD
ADVANTAGES OF CAD
• More efficient fragmentation
ADVANTAGES OF IRMPD• Fast• No blind spots in spectra• water loss from b-ions• Laser pulse can burn off
salts from sample
Similar y- and b- ions are produced from either technique.
FTMS Research Project :
1. Obtain a mixture of proteins.
2. Use electrospray ionization to introduce a complex mixture of proteins into FT-ICR.
3. Isolate individual protein ions and dissociate them to generate amino acid sequence information.
4. Use amino acid sequence information to identify proteins from a database.
Traditional Sequencing Strategy
PROTEIN SAMPLE
DIGESTION
HPLC SEPARATION
DISSOCIATION
SEQUENCE
Goal of FTMS Research
PROTEIN SAMPLE
DISSOCIATION
SEQUENCE
Research Strategy: Proof of Concept
• Reduce sample complexity, use only a single protein.
• Determine feasibility of obtaining useful sequence information.– must be consecutive amino acids.
– need a string of at least eight amino acids for unique identification.
800 900 1000 1100 1200 1300m/z
+11
+12+13+14
+15
% R
elat
ive
Abu
ndan
ce
100
0
+10
Charge State Distribution for APV-1
b739+
b738+
b10512+
[M+14H]14+
b10612+
b10613+
y375+
400 600 800 1000 1200m/z
0
100
% R
elat
ive
Abu
ndan
ceAPV-1 CAD MS2 of 85214+
Ac-- A M T D X X S A D D X K K A V G A F A
D K S K K K X G V M E F F K K H N F S
A AE
K A F H X X D K D R S G F X E E D E X
DV K
+
+
+
++
++
+
+ + +
X X A K T E K D S X D R A D P T F G K X
K S V
D K D G D G K X G V D E F T S X V T V S -- OH
VA G
APV-1 Product Ions from MS2 of 85214+
800 900 1000 1100 1200 1300m/z
+11
+12+13+14
+15
% R
elat
ive
Abu
ndan
ce
100
0
+10
Charge State Distribution for APV-1
400 800 1200 1600 2000
m/z
0
100
% A
bund
ance
y365+
b738+
b737+
b515+
b10812+
b10612+
[M+13H]13+
APV-1 CAD MS2 of 91813+
Ac-- A M T D X X S A D D X K K A V G A F A
D K S K K K X G V M E F F K K H N F S
A AE
K A F H X X D K D R S G F X E E D E X
DV K
+
+
+
++
+ ++
+
+
+
+X X A K T E K D S X D R A D P T F G K X
K S V
D K D G D G K X G V D E F T S X V T V S -- OH
VA G
APV-1 Product Ions from MS2 of 91813+
Identifying Proteins with FT-ICR ? ? ?
• Intact proteins can absorb energy without producing many fragments.
• Fragments are formed primarily at aspartic acid residues.
• Not enough sequence information is generated to identify proteins from databases.
O
NH
R 2O
NH
O
OH
Aspartic Acid Effect on Peptide Fragmentation
Protein Sequencing by Mass Spectrometry
PROTEIN SAMPLE
DISSOCIATION
SEQUENCE
DIGESTION
HPLC SEPARATION
NO!
Use MS3 capability of FT-ICR MS to obtain
protein sequence information.
Ubiquitin CAD MS2 of 78011+
b173+
y244+ y58
9+
y588+
b527+
b162+ b17
2+ b182+
[M+11H]11+
400 600 800 1000 1200m/z
0
100
%
Rel
ativ
e A
bund
ance
H - M Q I F V K T L T G K T I T L E V E P
Q Q D P P I G E K D Q I K A K V N E I
F A G K Q L E D G R T L S D Y N I Q K
HO -G G R L R L V L H L
S DT
RL I
E ST
+
+
++++
+
++
++
Ubiquitin Product Ions from MS2 of 78011+
H - M Q I F V K T L T G K T I T L E V E P
Q Q D P P I G E K D Q I K A K V N E I
F A G K Q L E D G R T L S D Y N I Q K
HO -G G R L R L V L H L
S DT
RL I
E ST
+
+
Ubiquitin Product Ions from MS3 of 78011+ 6824+
• Product ions (from MS2) with greatest abundance are large protein fragments that provide little new information using MS3.
• Smaller product ions have insufficient ion abundance for MS3.
• Product ions are still prone to aspartic acid effect.
Methyl Esterification of Peptides
CH3OH + H2O
H+
O
NH
O
OCH3
NH
O
NH
O
OH
NH
Product Ions from MS2 of 79511+
Methylated Ubiquitin
400 600 800 1000 1200m/z
0
100
% R
elat
ive
Abu
ndan
ce
b173+
y598+
y589+
y588+
b162+ b17
2+ b182+
[M+11H]11+
H - M Q I F V K T L T G K T I T L E V E P
Q Q D P P I G E K D Q I K A K V N E I
F A G K Q L E D G R T L S D Y N I Q K
HO -G G R L R L V L H L
S DT
RL I
E ST
+
++++
+
++ + +
Product Ions from MS2 of 79511+
Methylated Ubiquitin
400 600 800 1000 1200m/z
0
100
% A
bund
ance
y588+
y405+
y567+
y435+
y406+
y132+
b183+
Product Ions from MS3 of 78011+ 6824+
Methylated Ubiquitin
H - P
Q Q D P P I G E K D Q I K A K V N E I
F A G K Q L E D G R T L S D Y N I Q K
MeO -G G R L R L V L H L
S DT
RL I
E ST
+
++
+
+ +
+
Ubiquitin Product Ions from MS3 of 8358+
Methylated Ubiquitin
+++++++
+
H - M Q I F V K T L T G K T I T L E V E P
Q Q D P P I G E K D Q I K A K V N E I
F A G K Q L E D G R T L S D Y N I Q K
MeO -G G R L R L V L H L
S DT
RL I
E ST
+
++++
+
++ + +
+
++
+
+ +
+++++++
+
Product Ions from MS3 of Methylated Ubiquitin
Research Summary
• Aspartic acid modification may improve protein fragmentation by CAD.
• Sufficient amino acid sequence information can be obtained using FTMS to retrieve protein identification from a database.
Protein Sequencing by Mass Spectrometry
PROTEIN SAMPLE
CAD
SEQUENCE
DIGESTION
HPLC SEPARATION
NO!
Protein Sequencing by Mass Spectrometry
PROTEIN SAMPLE
DISSOCIATION
SEQUENCE
ChemicalModification
Proteomics
• Study of the PROTEin complement of the genOME.
• Nonexistent prior to 1995.
– Progress in genome sequencing research.
– Advances in mass spectrometry.
• Genome is static. Protein expression is dynamic.
– The presence of a gene does not guarantee protein expression.
– Proteins do the work of the cell.
Goal of Proteomics Research:
• Compare healthy and diseased tissue.• diagnose disease states.• develop new drug therapies.
• Determine effects of new pharmaceuticals.
• Cell differentiation, cell death.
• Provide insight into protein function.
Identify proteins that are expressed by a cell population
Challenges of Proteomic Research
• Cell extracts produce very complex mixtures of proteins.
• Even more complicated mixtures of peptide fragments from enzymatic digestion.
• Hundreds of peptides co-elute during a single HPLC run
• How can sequence information be obtained from each peptide ?!?
Proteomic Strategies
• Use two-dimensional gel separations to reduce sample complexity.
• Use mass spec. technology suited for collecting a large number of MS/MS spectra.
• Use database searching algorithms to identify protein sequences.
• Use peak parking.– Davis, M. T.; Stahl, D. C.; Hefta, S. A.; Lee, T. D. Anal. Chem.
1995, 67, 4549-4556. – Martin, S.E.; Shabanowitz, J.; Hunt,* D. F.; Marto, J.A ; Anal. Chem. 2000, 72, 4266-4274.
Proteomics and FTMS
• Advantages:– High sensitivity.
– High mass accuracy.
• Disadvantages:– FTMS software cannot operate ‘on the fly’.
– Extremely difficult to identify a precursor and construct and apply a SWIFT isolation waveform.
• Solution:– Perform the analysis in two sequential runs.
Mixture of Six Standard Proteins
Protein Moelcular Weight
(Da)
Solution Concentration
b-Casein 23623.35 1 x 10-6 M
Bovine Serum Albumin
69293.34 2 x 10-7 M
GA3PDH 35783.0 4 x 10-8 M
Carbonic Anhydrase (II)
28982.62 1.5 x 10-8 M
Beta-lactoglobulin 18281.34 4 x 10-9 M
Cytochrome C 12384 1 x 10-9 M
Chromatogram of ion current
Time (min)
0 1.0 2.0 3.0 4.0 5.00
50
100R
elat
ive
Abu
ndan
ce
Tryptic peptides derived from digesting a mixture of six proteins
A Single Mass Spectrum
600 800 1000 1200m/z
Rel
ativ
e A
bund
ance
*
Asterisk at mass 736 indicates an ion from Cytochrome C protein present in the mixture at 1x 1017 moles.
Approximately 25 ions are co-eluting. Proteins in mixture are present in 1000-fold concentration range
88 145 292 405 534 663 778 907 1020 1166 S G F X E E D E X K1166 1080 1022 875 762 633 504 389 260 147
bn
yn
250.0 500.0 750.0 1000.0
% A
bund
ance
100
0
y1
y2
y3
y4
y5
y6
y7
b9 / y8
b8
b7b6
b5
b4
b3
b2
[M+2H]2+
m/z
Proteomics Results
Protein
Number of Fragments
Found
Amino Acids Identified
Percent Coverage
b-Casein 7 70/209 33.5
BSA 17 152/607 25.0
GAPDH 24 298/335 89.0
CA II 9 92/259 35.5
BLG 9 66/162 40.7
Cyto C 7 58/104 55.7
Success with Unknown Mixtures of Proteins from Cells
• Peptide sequences were obtained from a complex mixture of proteins.
• The ultra-high mass accuracy improves confidence in protein assignments and decreases search times using computer database searching algorithms.
• Proteins were identified.
Results
Protein Candidate
Precursor m/z
(observed)
Precursor m/z
(predicted)Error (Da)
Number of product
ions present
Actin 895.950 895.949 0.001 16/30
GAPDH 788.406 788.397 0.008 9/26
Alpha Crystallin
583.333 583.333 0.000 8/20
P20 578.804 578.804 0.000 8/18
Acknowledgements
Doug Beusmann Tracie Bishop
Jennifer Caldwell Rob Christian
Scott Ficaro Erin Field
Leslie Frost Andy High
Gina King Jarrod Marto
Paul Russo Bob Settlage
Pam Thompson Forest White
Professor Donald F. HuntDr. Jeffrey Shabanowitz
