Proteomics at Indiana University

1
Proteomics at Indiana University
Randy J. Arnold Manager Proteomics Research
Development Facility Department of
Chemistry Indiana University, Bloomington
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What is Proteomics?
Proteomics is the study of protein expression,
regulation, modification, and function in living
systems for understanding how living systems use
proteins. Using a variety of techniques,
proteomics can be used to study how proteins
interact within a system, or how proteins change
due to applied stresses. Proteomics requires the
use of advanced measurement techniques including
separations, mass spectrometry, and advanced
imaging.
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The Central Dogma of Life
DNA genetic information
Replication
Transcription
RNA instructions for proteins
Translation
Proteins structure and function of living cells
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The Basics - Proteins
Polymers of amino acids (20 naturally occurring)
R1
H
H2O

H
C
CO2-
N
C
NH3
C
H
R2
O
R1 CH3 Alanine Ala A
R2 CH3OH Serine Ser S
AlanylSerine Ala-Ser AS
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The Basics Protein Structure
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Top-Down Proteomics
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Bottom-up Shotgun Proteomics
lyse cells
Mixture of 1000s of peptides
digest with trypsin
2-D LC-MS/MS
RPLC-MS/MS
LC-IMS-MS/MS
SCX steps
LC
LC
drift
m/z
m/z
Database searching - matching MS/MS data with
peptide sequence
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Chromatographic Separation

• Reverse-phase chromatography

5 mm
75 mm
100 Å pores
hydrophobic stationary phase
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Chromatographic Separation

• Molecular Interactions

NPLTGLAK
MILSGVAR
1) 95 aqueous / 5 organic mobile phase
2) 75 aqueous / 25 organic mobile phase
3) 65 aqueous / 35 organic mobile phase
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Electrospray Ionization

• 2002 Nobel Prize in Chemistry awarded to John
  Fenn for advancing electrospray

0 kV
2.5 kV




Gas-Phase Peptide ions
Hydrated Peptide ions
Peptide ions in droplets
Compliments of Dr. Myeong Hee Moon
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Mass Spectrometry

• Time-of-Flight MS

22 kV
1) Ions enter source region, accelerated toward
reflectron.
2) Ions separate in space based on their relative
mass-to-charge (m/z).
0 kV
0 kV
3) Ions reverse path in reflectron.
4) Ions impact detector.
0 kV
0 kV
H
L
M
20 kV
comp
amp
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Database Searching (Informatics)
b1
b2
b3
b4
b5
b6
b7
NH2
CO2
m/z
y7
y6
y5
y4
y3
y2
y1
Database searching software
Results
MASCOT

• Proteins found
• Hemoglobin, beta chain
• Pept. Mass Score Sequence
• 738.84 41 HLDNLK
• 912.01 61 VHLTDAEK
• 915.06 56 AAVNGLWGK
• 1090.24 41 VINAFNDGLK
• 1122.33 62 VVAGVASALAHK
• 1218.42 70 LVINAFNDGLK

Database (SwissProt) Actin MYTCVPIASEQUENCEMIMEWTP
QSDLIRPTVCIMNERCVGGPYILCMTEND Amylase DSLIKRNYTIPM
CSQIRECNHIPLMTRCHGYYKWSIALAINTQSFGIVRIVAMNKLPSSCRT
IVGHWEDRICTMQNCISPPEKELIAVARGTSP
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Faculty involved in Proteomics
David E. Clemmer Robert and  Marjorie Mann
Chairman and Chair Analytical Chemistry
Milos V. Novotny Distinguished Professor and
Lilly Chemistry Alumni ChairBioanalytical
Chemistry and Chemical Biology
James P. Reilly ProfessorPhysical and Analytical
Chemistry
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David Clemmer
Research Interests Ion mobility
spectrometry Protein structure measured in the
gas phase Multi-dimensional peptide separation /
mass analysis for proteomics and combinatorial
libraries Proteomics of fly aging Human urinary
proteome
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Clemmer Research - Instrumentation
http//www.indiana.edu/clemmer/
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Milos Novotny
Research Interests capillary electrophoresis /
electrochromatography electrospray ionization /
MALDI glycomics and glycobiology characterizatio
n of posttranslational modifications pheromone
isolation / identification
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Determination of Sites of Glycosylation in
Glycoproteins by QTOF MS
100
366.17
VENHTACHCSTCYYHK
3151.41
3516.64
3152.56
3517.54
2989.36
Hex

HexNAc
Hex
3808.75
3153.37
204.10
3518.78
PeptideHexNAc
3515.43
NeuAc
292.13
3441.52
2786.23
2988.52
1270.59
3354.58
2096.96
2624.18
657.30
1577.15
2787.33
3821.45
1173.85
3166.71
1496.80
2549.11
2079.99
731.38
1577.88
2098.08
Hex
1049.51
HexNAc
3529.69
Hex
Hex
HexNAc
1584.72
547.22
1759.86
3822.85
3645.67
820.33
968.60
mass
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
Y. Mechref and M. V. Novotny
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MALDI/TOF/CID Spectrum of N-Glycans Derived from
Ribonuclease B (Man5)
Y. Mechref, A. G. Baker, M. V. Novotny,
Carbohydrate Res., 313 (1998) 145155.
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Tandem MS of N-Glycans Derived from Ribonuclease
B (Man5)
Na
3,5X4a
100
22.9524
90
1,5X3a
3,5A5
3,5A4
3,5A3
1,5X4a
1,4A5
3,5X4a
80
2,4A5
0,4A3
70
3,5X3b
1,5X2
1,5X4a
B4
0,2A5
0,2A4
60
Intensity
1,5X3b
1036.3893
50
1,5X4x,3b
Ion D B3/Y3b
1257.5524
0,4A3
1,5X3a
40
C3
1,5X2
2,4A5
3,5A4
B3/Z3b
B2a
1123.4529
671.2255
Y2
30
849.2728
799.2855
(Hex)2
B4/Y4x,3b
475.1992
C2a
C1
C4
Y1
1095.4425
907.3265
447.1826
3,5A3
3,5X4x,3b
1,5X0
3,5A5
653.2201
0,2A5
Y3a
0,2A4
655.2078
Z2
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347.1320
Z1
527.2029
B3
1,5X1
1061.4415
B1
771.2791
874.3221
509.1753
550.2066
979.3621
429.1849
583.2186
625.2424
Y4x
226.0908
399.1900
689.2335
73.2773
1156.4673
164.9358
327.1572
306.8996
41.1236
244.1070
10
745.2795
953.3788
1010.3968
817.2909
1182.4816
365.1320
712.2309
272.1050
185.0653
147.7515
933.3752
607.1801
203.0858
91.4020
0
9.0
271.6
534.2
796.8
1059.4
1322.0
Mass (m/z)
Y. Mechref, M. V. Novotny, Cheni Krishnan Anal.
Chem. 2003, 75, 4895-4903.
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James Reilly
Research Interests MALDI-TOF mass spectrometry
of proteins MS fingerprinting of bacteria
proteins MALDI-TOF photo-induced dissociation
MS/MS Guanidination / MALDI peptide mass
mapping Characterization of surface-bound
biomolecules
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Guanidination
O
O
CH3
N
N
N
N
O

?
C
CH3OH
NH2
H2N
NH
NH2
O-methyl isourea
Lysine, K
Homoarginine
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Guanidination of Myoglobin Trypsin Digest
Intensity
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Ribosomes of E. coli
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Proteomics Research Development Facility
Milos V. Novotny, Director Randy J. Arnold,
Manager Yehia Mechref, Scientist Petra Hrncirova,
Post-doc Myeong Hee Moon, Visiting Scientist,
02-03 Chemistry grad students Bioinformatics
grad students
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Collaborations
IU School of Medicine William McBride
alcoholism / rat model Meei-Huey Jeng breast
cancer Linda Malkas breast ovarian cancer
Jian-Ting Zhang cancer / cell division Mark
Geobl yeast cell division IU Department of
Biology Peter Cherbas Drosophila
development Yves Brun bacteria cell cycle
control
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Future Directions

• New facilities
• Multidisciplinary Science Building, 2006
• New education initiatives
• Comprehensive Human Biology Program
• Masters in Biotechnology
• New technologies
• Separations
• Mass spectrometry

Chemistry
Myers Hall (Medical Sciences)
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Acknowledgments
Faculty Milos Novotny David Clemmer James Reilly
Collaborations William McBride Frank
Witzmann Meei-Huey Jeng
Scientists / Post-docs Yehia Mechref Myeong Hee
Moon Petra Hrncirova Steve Valentine Weidong
Cui Dariusz Janecki Wendy Strother-Robinson Li-Yun
Chang
Graduate students John Taraszka Rená
Sowell Ruwan Kurulung Matt Thompson Arugadoss
Devakumar Sunnie Myung Kiran Annaiah (informatics)
Resources Information Technology Group
Funding INGEN Indiana Genomics Initiative