(Bio)chemical Proteomics
Alex Kentsis
October, 2013
http://alexkentsis.net
A brief history of chemical
proteomics
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1907: Eduard Buchner, demonstration of cell-free alcohol fermentation (i.e.
enzymes)
1946: James Sumner, urease crystallization (enzymes can be pure proteins)
1970: Ulrich Laemmli, protein fractionation using SDS-PAGE
1972: Christian Anfinsen, renaturation of active ribonuclease (chemical
activity is related to protein conformation)
1973: Pedro Cuatrecasas with C. Anfinsen, purification of staphylococcal
nuclease using aminophenyl solid support affinity chromatography (based
on Lerman’s purification of tyrosinase by aminophenol in 1963 and
Starkenstein’s purification of amylase using starch in 1910)
1973, Hugh Niall, “protein sequanator” for automated identification of
proteins (based on Pehr Edman’s amino acid cleavage in 1950)
1986, Don Hunt, protein sequencing using tandem mass spectrometry
(based on Keith Jennings’ collision-induced dissociation)
1996, Marc Wilkins, “proteomics” using 2D electrophoresis and database
matching
…
Mass spectrometry
Thomson JJ (1913) Rays of positive electricity,
Proceedings of the Royal Society, A 89, 1-20
Dempster AJ (1918) A New Method of Positive Ray
Analysis, Phys. Rev. 11 (4): 316–325 (discovery of 235U)
Bainbridge KT (1933) The Equivalence of Mass and
Energy, Phys. Rev. 44 (2): 123. (E = mc2)
Lawrence EO (1945) Calutron separation of 235U
(Manhattan Project)
Beckey HD (1969) Field ionization mass spectrometry
Research/Development 20 (11): 26.
(Soft ionization that preserves chemical structure)
Mass spectrometry
Mass spectrometry
• What is required?
– Ionization and transfer of molecules into gas
phase
– Ion separation and detection based on
mobility in vacuum in EM field
Ionization methods
Ionization Source
Acronym
Event
Electrospray ionization
ESI
evaporation of charged
droplets
Atmospheric pressure
chemical ionization
APCI
corona discharge and
proton transfer
Matrix-assisted laser
desorption/ionization
MALDI
photon absorption/proton
transfer
Desorption/ionization on
silicon
DIOS
photon absorption/proton
transfer
Fast atom/ion
bombardment
FAB
ion desorption/proton
transfer
Electron ionization
EI
electron beam/electron
transfer
Chemical ionization
CI
proton transfer
Electrospray ionization
+-
+-
+
+
+
+
+
+
+
Surface tension
vs.
Electrostatic repulsion
+
+
+ + +
+ + +
Taylor cone
“Electrospray wings for molecular elephants”
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2002/fenn-lecture.html
Electrospray ionization
+
+
+
+ +
+
+ ++
+
+
+
+ + +
+
+
+
+
+
+
Solvent
evaporation
+ +
+
++
Coulombic
explosion
+ +
Increasing Charge Density
http://pubs.acs.org/doi/abs/10.1021/ac00070a001
Mass analyzers
Mass Analyzers
Event
Magnetic Sector
magnetic field affects radius of curvature of ions
Time-of-Flight (TOF)
time-of-flight correlated directly to ion's m/z
Quadrupole
scan radio frequency field
Ion Trap
scan radio frequency field
Fourier Transform Ion Cyclotron Resonance MS
translates ion cyclotron motion to m/z (FTMS)
Ion detectors
TOF, Ion traps
FTICR, Orbitrap
R Voltage across
resistor
Single ion detection
Ions “image current” detection
Mass spectral interpretation
Mass spectral resolution
Peptide sequencing using tandem
MS
Collisionally-Activated Dissociation or
Collision-Induced Dissociation
• The molecules are accelerated into a cell filled with
an inert collision gas (Ar, He, etc)
• Molecules undergo multiple collision, accumulating
energy until they undergo chemical dissociation
• The peptide bond and weak side chain bonds are
predominantly cleaved
Peptide sequencing using tandem
MS
y
7
O
y
6
y
5
R
2
O
y
4
R
4
O
H
N
HN
2
O
a
2
b
2
R
3
O
b
4
R
8
H
N
OH
N
H
O
b
3
y
1
R
6
N
H
R
1
y
2
H
N
N
H
b
1
y
3
R
5
N
H
O
b
5
Roepstorff-Fohlmann-Biemann-Nomenclature
b
6
R
7
O
b
7
The CID Mechanism
Charge-induced
fragmentation
Peptide sequencing using tandem
CID
986.593
1244.702
1115.644
899.013
E
129
E
129
1343.766
V
99
V
1442.865
99
Peptide sequencing using tandem
CID
Monoisotopic Mass
Glycine
Alanine
Serine
Proline
Valine
Threonine
Cysteine
Isoleucine
Leucine
Asparagine
57.02147
71.03712
87.03203
97.05277
99.06842
101.04768
103.00919
113.08407
113.08407
114.04293
Aspartic acid
Glutamine
Lysine
Glutamic acid
Methionine
Histidine
Phenylalanine
Arginine
Tyrosine
Tryptophan
115.02695
128.05858
128.09497
129.0426
131.04049
137.05891
147.06842
156.10112
163.06333
186.07932
Protein identification using tandem
MS
C
A 100
Relative Intensity (%)
[M+2H]2+
642.5
0
T
CATTAAACTAAAAAGATGTCCCTATATGATGACCTGGGAGTGGAGACCAGTGACTCAA
AAACTGAAGGCTGGTCCAAAAACTTCAAGCTCCTGCAGTCCCAGCTCCAGGTGAAGAA
GGCGGCGCTCACTCAGGCCAAGAGCCAAAGGACCAAGCAAAGTACAGTGCTTGCTCCG
GTCATCGACCTAAAGCGAGGCGGCTCCTCAGATGACCGGCAGATTGCAGACACACCAC
CTCACGTGGCAGCTGGGCTGAAGGACCCTGTGCCCAGTGGGTTTTCTGCAGGGGAAGT
TCTGATTCCCTTAGCTGA
T
400
500
600
m/z
700
800
Relative Intensity (%)
[M+2H]2+
642.5
MS/MS
A I/L V
T
1068.6
400
600
m/z
Translation into first reading frame
H*TKKMSLYDDLGVETSDSKTEGWSKNFKLLQSQL
QVKKAALTQAKSQRTKQSTVLAPVIDLKRGGSSDD
RQIADTPPHVAAGLKDPVPSGFSAGEVLIPLA
Cloning
684.4
200
Peptid sequence tag: (684.4)ALVT(1068.6)
m=1283.0 Da
data base search
EST-Sequence (ID:404211)
TT
T
B 100
0
MS
T
800
1000
1200
Novel protein: SPF45
SLYDDLGVETSDSKTEGWSKNFKLLQSQLQVKKAALTQAKSQRTKQSTVLAPVIDLKR
GGSSDDRQIADTPPHVAAGLKDPVPSGFSAGEVLIPLADEYDPMFPNDYEKVVKRQRE
ERQRQRELERQKEIEEREKRRKDRHEASGFSRRPDPDSDEDEDYERERRKRSMGGAAI
APPTSLVEKDKELPRDFPYEEDSRPRSQSSKAAIPPPVYEEPDRPRSPTGPSNSFLAN
MGGTVAHKIMQKYGFREGQGLGKHEQGLSTALSVEKTSKRGGKIIVGDATEKGEAQDA
SKKSDSNPLTEILKCPTKVVLLRNMVGAGEVDEDLEVETKEECEKYGKVGKCVIFEIP
GAPDDEAVRIFLEFERVESAIKAVVDLNGRYFGGRVVKACFYNLDKFRVLDLAEQV
Protein identification using tandem
MS
Biological mass spectrometry
• Molecule fractionation compatible with soft
ionization on mass spectrometric time-scales
– Reverse phase LC-MS with volatile solvents
– Reduction of spectral complexity and
miniaturization improve sensitivity
LC-MS
LC
Column
Soft ion source
Mass analyzer
Detector
High-resolution tandem MS
High-resolution parallel MS
Dual-pressure
linear ion trap
Ultra-high-field
Orbitrap mass analyzer
Ion-routing multipole
Quadrupole mass filter
Active beam
guide (ABG)
EASY-ETD ion source
The One Hour Yeast Proteome
http://www.ncbi.nlm.nih.gov/pubmed/24143002
Quantitative mass spectrometry
Relative Abundance
Stable isotope detection using high-resolution mass spectrometry
C12 98.9 %, C13 1.10 %
N14 99.63 %, N15 0.37 %
H1 99.985 %, H2 0.015 %
O16 99.76 %, O17 0.04 %, O18 0.20 %
50
Δ1 Da
Δ2 Da
0
Quantitative mass spectrometry
100
Lysine (heavy)
Elution profile
Relative Abundance
Lysine (light)
0
18.4
18.8
19.2
19.6
20.0
20.4
Time (min)
489.77
Δm = +8 Da
Δm/z = 4 Da
100
490.27
493.77
494.27
490.77
493.27
Δm = +8 Da
494.77
491.77
0
489.5
490.5
491.5
[ASVLFANEK]2+
492.5
493.5
494.5
495.5
[ASVLFANEK]2+
Ultrahigh-resolution quantitative
mass spectrometry
http://www.nature.com/nmeth/journal/v10/n4/full/nmeth.2378.html
Activity-based protein profiling
Given chemical probe, can identify enzymatically active targets
Considerations
Specific vs. pleiotropic
Covalent vs non-covalent
Chemically and sterically permissive functional group
http://dx.doi.org/10.1016/j.cbpa.2003.11.004
Quantitative mass spectrometry for
chemical profiling
http://www.pnas.org/content/106/12/4617.abstract
Quantitative mass spectrometry for
chemical profiling
Specificity of molecular interactions
depends on concentration, e.g.
all drugs are pleiotropic
Biological macromolecules exist
as non-covalent complexes
Chemical specificity is not directly
related to biological activity
Affinity-based proteomics reveal
cancer-specific networks
coordinated by Hsp90
http://www.nature.com/nchembio/journal/v7/n11/full/nchembio.670.html
Proteome-wide drug binding
analysis
http://www.nature.com/nbt/journal/v29/n3/full/nbt.1759.html
Chemical proteomics of lysine
deacetylases
Chemical proteomic profiling using
drug libraries (kinobeads)
http://www.nature.com/nbt/journal/v25/n9/full/nbt1328.html
Towards functional chemical
proteomics
http://www.ncbi.nlm.nih.gov/pubmed/18662549
Modern biological mass
spectrometry
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Structural characterization
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Elemental composition from high-accuracy mass measurements
Chemical structure from gas phase fragmentation reactions http://chemdata.nist.gov
Peptide sequencing using amino acid dissociation
Supramolecular complex structure using soft ionization
Quantitative analysis
– High sensitivity (zeptomole) and specificity (gold standard) ensured by direct ion
detection, and selected ion and reaction monitoring (SIM and SRM) techniques
– Metabolites (FDA, NBS)
– Genotyping (Sequenom)
– Molecular biomarkers (Advion)
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Functional profiling
– Chemical activity using affinity chromatography coupled to MS
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Activity-based protein profiling (ABPP)
Activity correlation proteomics
– Functional activity using stable isotope labeling of biological processes
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Stable isotope labeling in culture (SILAC)
…
Mountain of biochemical knowledge
?
Chemistry
Enzymology
Structural analysis
Chemical mechanism
Metabolism
Cell growth and development
Pleiotropy
Biological function
Reproduction and use
• Attribution
– This presentation may contain photographs and
images from other sources, which are referenced
or tagged electronically
• Academic use
– You may freely use this material for academic
purposes as long as you properly acknowledge it
• Commercial use
– Please contact Alex Kentsis for commercial use