Proteomics via Mass Spectrometry (a bioinformatics perspective)
Mass Spectrometry and Proteomics Paolo Lecchi, PhD Dept. of Pharmacology George Washington...
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Transcript of Mass Spectrometry and Proteomics Paolo Lecchi, PhD Dept. of Pharmacology George Washington...
![Page 1: Mass Spectrometry and Proteomics Paolo Lecchi, PhD Dept. of Pharmacology George Washington University October 13, 2003.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649e125503460f94afe37c/html5/thumbnails/1.jpg)
Mass Spectrometry and Proteomics
Paolo Lecchi, PhDDept. of Pharmacology
George Washington University
October 13, 2003
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WHAT IS A “MASS SPECTROMETER ”...?
MS
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The black box problem…...
ESI MALDI LC/MS LC-MSMSESI
ESI Ion trap TOF QTOFquad.
ESI CRIMS EI APCIFAB
FT-ICR
qQTOF
SELDI
…many black boxes !
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“A MASS SPECTROMETER MEASURES THE MW….”
“...A MS ANALYSIS GIVES THE MASS-TO-CHARGE RATIO (m/z)
OF IONS…IN GAS PHASE”.
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Data Processing
ION SOURCE
ANALYZER ion separation
vacuum
Detector
Pumping system
Sample introduction
DIRECT INTRODUCTION
(solid, liquid, gas) SEPARATION
TECHNIQUE (HPLC, CE, GC)
MALDI, FAB, EI, ELECTROSPRAY
TOF, quadrupole, ION TRAP
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ESILiquid flow
Q or Ion Trapanalyzer
ESI is a solution technique that gives a continuous stream of ions, best for quadrupoles, ion traps, etc.
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MALDI3 nS LASER PULSE
Sample (solid) on target at high voltage/ high vacuum
MALDI is a solid-state technique that gives ions in pulses, best suited to time-of-flight MS.
TOF analyzer
Atmosphere Low vac. High vac.
High vacuum
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….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)
Until recently only ESI was available for high quality tandem-MS
MALDI is easier to use and maintain
MALDI is more “flexible” (MW from 200 to 400,000 Da)
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0
20
40
60
80
100
%Int.
5000 10000 15000 20000Mass/Charge
Data: 1015pl30001.O7 15 Oct 2002 16:22 Cal: 16 Oct 2002 8:12
8480.6
5653.4
16953.04239.4
MALDI-TOF spectra of apomyoglobin
INSTRUMENT: Kratos Axima-CFR
Sample: 1 pmole apomyoglobin (horse skeletal muscle)
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400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
+101695.9
+111541.8
+121413.5
+131304.8
+141211.7
+15
1131.0
+16
1060.4
+17
998.1
+18
942.8
+19893.2
+20848.6
+21808.2
+22771.5
+23738.0+24
707.4+25
679.1
+91884.5
ACTUAL SPECTRUM
+91884.3
16000 16400 16800 17200 17600 18000 18400
mass
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
16951.5
AFTER DECONVOLUTION
ESI-ion trap spectra of apomyoglobin
INSTRUMENT: Thermoquest LCQ-classic
Sample: 1 pmole apomyoglobin (horse skeletal muscle)
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A “RESEARCH GRADE” MS (200 to 500 k$) PROVIDES AN ACCURATE MW DETERMINATION:
~ 10 ppm (e.g. 1000.0 ± 0.1)
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ACCURACY IS NOT THE ONLY PARAMETER TO BE CONSIDERED IN A MASS
SPECTROMETER...
...don’t forget the “S factors”
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Sensitivity = femtomole 10-15 M (...attomole 10-18 M)
Simplicity = very easy training required
$$$ = 70 to 650 k$ 120 to 650 k$
Speed (high throughput)= ~104/day dynamic system
Structural information = MS/MS MSn
Software = “ ...evaluation in progress.”
MALDI ESI
Selectivity (resolution) = >5000
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%Int.
2092 2094 2096 2098 2100 2102 2104 2106 2108Mass/Charge
Data: NDR_thin_2Ghz_blk_6fload0001.G24 20 May 2002 16:49 Cal: Kent_SP2_blk_1567 20 May 2002 14:31 Kratos PC Axima CFR V2.3.0a: Mode reflectron_2GHz, Power: 48, Blanked, P.Ext. @ 2095 (bin 135)
2099.17
2098.18
2100.18
2101.19
MALDI analysis of a peptide m/z 2098 (6 fmoles loaded)
Expected peptide mass 2098.20
(0.02 amu difference) Accuracy: (~10 ppm)
0.1 amu
resolution: ~20,000
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NEW INSTRUMENT DESIGNS MAKE ESI AND MALDI MORE “INTERCHANGEABLE”...
Orthogonal acceleration-TOF is compatible with a continuous ion current, such as from ESI.
Liquid
Pusher electrode with pulsed VAcc
To reflectron TOF
Needle@4kV ions
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N2 Laser
Ar collision gas
Hinged door
96-well MALDI target
1x10-2 Torr
2x10-5 Torr
5x10-7
Torr
Liner
Ion mirror
MCP
PusherPuller
Q1 q2
MS1 mass
selection
CID
MS2 mass selection
Thermal equilib.
q
0
UV laser pulses via attenuator,
fiber optic cable and lens to
vacuum system
Schematic of QSTAR (with MALDI source)
MALDI can be carried out at “high” pressure. Ions collide with gas molecules, are slowed down and thermally equilibrated. Pulses are “smeared out” to give a semi-continuous ion current.
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“...for their developments of soft desorption ionisation methods for mass spectrometric analysis of biological macromolecules”.
Nobel Prize in Chemistry 2002
1//2 of the prize went to Kurt Wutrich (Switzerland) development of NMR analysis
1/4 to John B. Fenn (USA)
Virginia Commonwealth University
1/4 to Koichi Tanaka (Japan)
Shimadzu. Corp. Kyoto
Electrospray Laser Ionization
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“..other than biochemistry done very, very fast, is not entirely clear to me what proteomics is.”
Marvin Cassman, Director of the National Institute of General Medical Sciences *
C&E News, March 18, 2002
(*) ~90% of the NIH-funded mass spectrometry lab receive grants from NIGMS.
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DIFFERENTIAL EXPRESSION AND QUANTITATION
THE ROLE OF MASS SPECTROMETRY IN PROTEOMICS:
IDENTIFICATION OF PROTEINS, USUALLY IN COMPLEX MIXTURES
ANALYSIS OF POSTRANSLATIONAL AND CHEMICAL MODIFICATIONS
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Run 2Dgel; stain;
scan
Excise spot;elute; digest
1000 1500 2000 Mass (m/z)
Extract peptides;mass analyze
Protein identification
2D-PAGE AND MASS PECTROMETRY...…A PARADIGM IN PROTEOMICS
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SEPARATION electrophoresis (1-D, 2-D)
chromatography (SEC, ion exchange, reversed phase)
DIGESTION chemical (BrCN)
enzymatic (trypsin,, Lys-C, Asp-C) reduction (Di-Thio-Threitol, -Mercapto-Ethanol) alkylation (IodoAcAcid, IodoAcAmide, Vynil Pyridine)
MALDI MS ANALYSIS
protein identification (peptide mass fingerprinting)
peptide structural information (post source decay)
EXPERIMENTAL PROCEDURES IN PROTEOMICS
SAMPLE CLEAN UP chromatography (reversed phase)
solid phase extraction (Zip Tip)
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Mass spectrometry improved substantially during the last 10 years...
2D-PAGE still is the most powerful separation technique but has several disadvantages...
2-D PAGE AND MASS SPECTROMETRY... ...NOT THE IDEAL TECHNIQUE FOR PROTEOMICS:
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Restricted to proteins < 106 and > 104 Da MW Cannot detect proteins expressed at low levels Limited to 600~800 separate spots Gel to gel reproducibility is poor Quantitation is poor, ± 50% or worse Dynamic range is limited, < 10X Analysis is not directly coupled to separation
DISADVANTAGES OF 2-D PAGE...
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MULTI-DIMENSIONAL SEPARATIONS: AN ALTERNATIVE TO 2-D
PAGEIn multidimensional chromatography two (or more) techniques with “orthogonal” properties are combined to achieve higher separation power.
FIRST DIMENSION: e.g. Size Exclusion, Ion-Exchange
SECOND DIMENSION: e.g. Reversed Phase Ion-Exchange
MS
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20 25 30 35 40 45 50 55
RETENTION TIME
1
2
3
4
5
6
7
8
9
10
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RO
TOFO
R F
RA
CTI
ON
NU
MB
ER
0.37|-|0.4
0.34|-|0.37
0.31|-|0.34
0.28|-|0.31
0.25|-|0.28
0.23|-|0.25
0.2|-|0.23
0.17|-|0.2
0.14|-|0.17
0.11|-|0.14
0.08|-|0.11
0.05|-|0.08
HYBRID 2-D SEPARATION: IEF-HPLC
Lecchi et. al. JBBM june 2003
Sample: E. coli extract
First dimension: y-axes IEF (Biorad rotofor)
Second dimension: x-axes IEF fractions separated by HPLC (reversed phase C-18)
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…to improve the efficiency of the proteolytic digestion it is important to reduce and alkylate disulfide bonds
s s
ss
ss
ss
s
ss
s
s
s s
RR
R
R
R
R
reduction alkylation
enzymatic digestion
R
Rs
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Enzymatic digestion
*Some proteolytic enzymes are very specific, e.g.:trypsin cuts only at Lys-X or Arg-XLys-C Lys-XArg-C Arg-XGlu-C (V-8) Glu-X and Asp-X
peptide bond
R-C-OH
O
H2OProteolytic*
enzyme
H
R-C N-R’
O
N-R’
H
H
+
protein
(proteolytic fragments)
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Mass/Charge
10 mV Profiles 1-50 Smooth Sv-Gl 2 -Baseline 20
Kratos PC Axima CFRplus V2.3.0: Mode reflectron, Power: 50, P.Ext. @ 1500 (bin 157)
14
28
.85
10
45
.68
87
4.5
3
17
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.00
10
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.68
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.99
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.22
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6.5
1
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.97
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6.0
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93
6.4
8
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.65
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.76
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.22
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53
.67
73
7.9
9
93
7.4
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66
6.0
4
10
31
.70
12
69
.76
18
06
.06
21
63
.26
22
74
.28
18
62
.99
Peptide Mass Fingerprinting (PMF)
MS analysis of proteolytic fragments is a common way to identify a protein.
Low MW peaks (e.g. < 500 Da) are not generally used because of the high
interference of the matrix.
The following masses are entered in a database for protein identification: 737.99 - 874.44 - 936.48 - 1030.65 - 1047.06 - 1153.67 - 1269.76 - 1428.85 1536.76 - 1676.97 - 1808.06 - 1862.99 - 2163.26 - 2274.28
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Database search for protein identification
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“IS NOT JUST ABOUT ‘SEPARATION POWER”
AN IDEAL METHOD SHOULD BE ABLE TO IDENTIFY AND QUANTIFY PROTEINS WHOSE EXPRESSION LEVELS CHANGE
GENES 35,000
PROTEINS 200,000
TRYPTIC PEPTIDES 4,000,000
THERE IS A HUGE DYNAMIC RANGE OF PROTEIN EXPRESSION (12 orders of magnitude)
1 peptide/sec = ~46 days
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“Proteomics is knowing the structure and function of all proteins from all organisms…that is not possible. We need to be more selective”.
George Kenyon, University of Michigan.
In his opening remark at the meeting: “Defining the Mandate of Proteomics in the Post-Genomic Era”.
National Academies, Washington DC, March 2002.