Mass Spectrometry and Proteomics

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Mass Spectrometry and Proteomics

• Paolo Lecchi, PhD
• Dept. of Pharmacology
• George Washington University

October 13, 2003
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WHAT IS A MASS SPECTROMETER ...?
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The black box problem...
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A MASS SPECTROMETER MEASURES THE MW.
...A MS ANALYSIS GIVES THE
MASS-TO-CHARGE RATIO (m/z) OF IONSIN GAS PHASE.
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3 nS LASER PULSE
MALDI
TOF analyzer
Sample (solid) on target at high voltage/ high
vacuum
High vacuum
MALDI is a solid-state technique that gives ions
in pulses, best suited to time-of-flight MS.
ESI
Liquid flow
Q or Ion Trap analyzer
Atmosphere Low vac. High vac.
ESI is a solution technique that gives a
continuous stream of ions, best for quadrupoles,
ion traps, etc.
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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)
MALDI is more flexible (MW from 200 to 400,000
Da)
Until recently only ESI was available for high
quality tandem-MS
MALDI is easier to use and maintain
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MALDI-TOF spectra of apomyoglobin
Sample 1 pmole apomyoglobin (horse skeletal
muscle)
INSTRUMENT Kratos Axima-CFR
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ESI-ion trap spectra of apomyoglobin
ACTUAL SPECTRUM
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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...
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MALDI ESI
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
Selectivity (resolution) gt5000
Structural information MS/MS
MSn
Software ...evaluation in
progress.
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MALDI analysis of a peptide m/z 2098 (6 fmoles
loaded)
Data NDR_thin_2Ghz_blk_6fload0001.G24 20 May
2002 1649 Cal Kent_SP2_blk_1567 20 May 2002
1431
Kratos PC Axima CFR V2.3.0a Mode
reflectron_2GHz, Power 48, Blanked, P.Ext. _at_
2095 (bin 135)
Int.
2099.17
100
90
2098.18
Expected peptide mass 2098.20 (0.02 amu
difference) Accuracy (10 ppm)
80
70
60
2100.18
50
40
30
2101.19
20
10
0
2092
2094
2096
2098
2100
2102
2104
2106
2108
Mass/Charge
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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.
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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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Nobel Prize in Chemistry 2002
...for their developments of soft desorption
ionisation methods for mass spectrometric
analysis of biological macromolecules.
1/4 to John B. Fenn (USA) Virginia Commonwealth
University
1/4 to Koichi Tanaka (Japan) Shimadzu. Corp. Kyoto
Electrospray
Laser Ionization
1//2 of the prize went to Kurt Wutrich
(Switzerland) development of NMR analysis
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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 CE News,
March 18, 2002
() 90 of the NIH-funded mass spectrometry lab
receive grants from NIGMS.
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THE ROLE OF MASS SPECTROMETRY IN PROTEOMICS
IDENTIFICATION OF PROTEINS, USUALLY IN COMPLEX
MIXTURES
ANALYSIS OF POSTRANSLATIONAL AND CHEMICAL
MODIFICATIONS
DIFFERENTIAL EXPRESSION AND QUANTITATION
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2D-PAGE AND MASS PECTROMETRY...A PARADIGM IN
PROTEOMICS
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EXPERIMENTAL PROCEDURES IN PROTEOMICS

• 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,
  b-Mercapto-Ethanol)
• alkylation (IodoAcAcid, IodoAcAmide,
  Vynil Pyridine)

• SAMPLE CLEAN UP chromatography (reversed phase)

solid phase extraction (Zip Tip)

• MALDI MS ANALYSIS
• protein identification (peptide mass
  fingerprinting)
• peptide structural information (post source
  decay)

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2-D PAGE AND MASS SPECTROMETRY... ...NOT THE
IDEAL TECHNIQUE FOR PROTEOMICS
Mass spectrometry improved substantially during
the last 10 years...
2D-PAGE still is the most powerful separation
technique but has several disadvantages...
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DISADVANTAGES OF 2-D PAGE...
? Restricted to proteins lt 106 and gt 104 Da MW ?
Cannot detect proteins expressed at low levels ?
Limited to 600800 separate spots ? Gel to gel
reproducibility is poor ? Quantitation is poor,
50 or worse ? Dynamic range is limited, lt
10X ? Analysis is not directly coupled to
separation
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MULTI-DIMENSIONAL SEPARATIONS
AN ALTERNATIVE TO 2-D PAGE
In multidimensional chromatography two (or more)
techniques with orthogonal properties are
combined to achieve higher separation power.
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HYBRID 2-D SEPARATION IEF-HPLC
Sample E. coli extract First dimension y-axes
IEF (Biorad rotofor) Second dimension
x-axes IEF fractions separated by HPLC
(reversed phase C-18)
Lecchi et. al. JBBM june 2003
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to improve the efficiency of the proteolytic
digestion it is important to reduce and alkylate
disulfide bonds
s
s
s
s
R
R
s
R
s
s
s
R
alkylation
reduction
s
s
s
R
R
s
s
R
s
R
s
s
enzymatic digestion
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Enzymatic digestion
Some proteolytic enzymes are very specific,
e.g. trypsin cuts only at Lys-X or Arg-X Lys-C
Lys-X Arg-C Arg-X Glu-C (V-8) Glu-X and
Asp-X
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Peptide Mass Fingerprinting (PMF)
MS analysis of proteolytic fragments is a common
way to identify a protein.
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
Low MW peaks (e.g. lt 500 Da) are not generally
used because of the high interference of the
matrix.
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Database search for protein identification
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IS NOT JUST ABOUT SEPARATION POWER
THERE IS A HUGE DYNAMIC RANGE OF PROTEIN
EXPRESSION (12 orders of magnitude)
AN IDEAL METHOD SHOULD BE ABLE TO IDENTIFY AND
QUANTIFY PROTEINS WHOSE EXPRESSION LEVELS CHANGE
1 peptide/sec 46 days
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Proteomics is knowing the structure and function
of all proteins from all organismsthat 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.