college biotechnologie

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A Roadmap for Mass Spectrometry Analysis of
Proteomics and MetabolomicsDR. SYED GHULAM
MUSHARRAFASSOCIATE PROFESSORHEJ RESEARCFJH
INSTITUTE OF CHEMISTRY UNIVERSITY OF KARACHI
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Are You on Right Track?
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The Omic Sciences side by side comparison
Journal of Surgical Oncology Volume 103,Issue 5,
pages 451-459, 28, 2011
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Proteomics
Branch of discovery science focusing on
proteins In 1994 the proteome was defined as
the complete set of proteins that is expressed
and modified following expression by the entire
genome in the lifetime of a cell. Today
proteomics is a scientific discipline that will
bridge the gap between our understanding of
genome sequences and cellular behavior.
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Scope of Proteomics
Global Entire proteome
Targeted individual proteins or a subset of the
entire proteome

• Identification of proteins
• Post-translational modifications (PTMs)
• Protein structure
• Localization mapping of proteins
• Protein/protein complexes and Interactions
• Protein expression in normal, stressed and
  diseases states
• protein markers for diagnostic purposes
• novel molecular targets for drug discovery
• Protein function in normal, stressed and
  diseases states

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Proteomic challenges
Protein size from 50 to 100,000 amino acids
/ 5,000-1,000,000 Da Relative abundance
broad dynamic range 10-1,000,000 copies /per
cell Diversity in human cells gt 30,000
different sequences Different forms
Post-translational modifications
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Proteomic challenges
Albumin, IgGs
Plasma proteins (transferrin, lipoproteins, hapt
oglobin, etc.)
Protein
Cytokines / chemokines, etc.
of proteins
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Protein Structure Primary sequence
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Protein Structure Primary sequence
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Protein Structure Folding into a conformation

• Proteins are self-assembling all of the
  information necessary to determine the final 3-D
  structure of the molecule is encoded in its
  sequence

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Complexity of the proteome
What can happen
What might happen
What is likely going to happen
What is happening
Swammerdam Institute for Life Sciences
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Mass spectrometer Principle
Sample
Ion Source
Desorp molecules as ions from a solid or solution
into the vacuum of the mass spectrometer
Mass analyzer
Separate the ions based on their m/z ratio
Mass spectrometer
Vacuum
Ion detector
Detect the mass separated ions
Construct a mass spectrum of the detected ions
Computer datasystem
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Tandem MS MS/MS
Sample
Sample
Ion Source
Ion Source
Mass analyzer
Mass analyzer
Ion fragmentation
Mass spectrometer
Mass spectrometer
Vacuum
Vacuum
Mass analyzer
Ion detector
Ion detector
Computer datasystem
Computer datasystem
MS spectrum
MSMS spectrum
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Mass spectrum
Mass range m/z detection range Isotopes the
molecules show an isotope m/z distribution Mass
Accuracy the ability to measure m/z of molecules
with high precision Mass resolution the ability
to individually detect molecules with closely
spaced masses Dynamic range the ratio of the most
over the least abundant molecules
detected Sensitivity the minimum amount of
detectable sample
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Mass Spectrometry and Life Sciences

• Development of suitable ionisation techniques to
  transfer biomolecules as ions into the gas phase
  (1988-1989)
• Sensitive detection and accurate mass
  determination of proteins, peptides,
  carbohydrates, glycoconjugates and nucleic acids
• Ionisation methods
• matrix-assisted laser desorption/ionisation
  (MALDI)
• electrospray ionisation (ESI)

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Steps in MALDI Sample Preparation
sample solution
matrix
analyte
insert target and perform analysis
dry samples
sample deposition
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MALDI matrices
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MALDI matrices
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Matrix-Assisted Laser Desorption Ionization
(MALDI)
Matrix for Proteins sinapinic acid,
dihydroxybenzoic acid etc. Matrix for Peptides
4-hydroxy-a-cyanocinnamic acid, DHB it
co-crystallizes, absorbs laser energy, evaporates
and acts as acid
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Principle MALDI-TOF-MS
Int
Mass spectrum
Laser
m/z
Detector
Clock
Sample Target
Molecular Mass
Time-of-Flight
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Electrospray ionisation
Electrospray Ionisation (ESI)
Coulomb Fission or Ion Evaporation
Coulomb Fission
Charge Separation
0.1-100 ?L/min
TaylorCone
Mass Spectrometer
Excesscharge on surface
Pltlt1 Atm
Sprayneedle2-5 KV
Drying gas Pressure 1 Atm
Spray current
Spray current
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A Roadmap for mass spectrometry analysis of
proteins
Isolation of proteins from tissue/cells
Purification
Digestion Of the protein mixture
HPLC separation of digest peptides
Peptide MS/MS data acquisition
Peptide fragment fingerprint
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Stategy identification
Isolation of proteins from tissue/cells
In-gel digestion approach
Schotgun approach
Purification
Gel electroforeses separation of proteins
Digestion Of the protein mixture
HPLC separation of digest peptides
In-gel digestion of spot-picked proteins
Peptide MS/MS data acquisition
Peptide MS data acquisition
Peptide Mass Fingerprinting
MS/MS ion search
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Proteome analysis using two-dimensional gel
electrophoresis and mass spectrometry (2DE/MS)
a major goal of proteomics is the global and
quantitative measurement of the proteins
expressed in cells or tissues
the qualitative and quantitative comparison of
proteomes under different conditions to further
unravel biological processes
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ESI MS of Macromolecules
ESI Spectrum of Trypsinogen (MW 23983)
M 15 H
1599.8
M 16 H
M 14 H
1499.9
1714.1
M 13 H
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio
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An Electrospray Investigation on in vitro
Glycation of Protein
Deconvoluted spectrum of BSA
ESI Spectrum of BSA
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An Electrospray Investigation on in vitro
Glycation of Protein
Mass of BSA Mass of glycated BSA Differences Added glucose molecules
66438 66761 323 2
  66921 483 3
  67082 644 4
  67246 808 5
  67409 971 6
  67562 1124 7
Glucose Concentration 0.2M Incubation Days 7
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An Electrospray Investigation on in vitro
Glycation of Protein
Glucose
Glucose molecules added within a week
ESI-Deconvoluted spectrum of glycated BSA
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ESI MS of Macromolecules
ESI Spectrum of Trypsinogen (MW 23983)
M 15 H
1599.8
M 16 H
M 14 H
1499.9
1714.1
M 13 H
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio
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MS Protein identification and database searching

• Preparation of a proteolytic digest of the
  protein, usually by trypsin (cleaves at C-side of
  K (lysine) and R (arginine), except KP and RP
• Determination of mass spectrum of peptide
  mixture experimental mass list
• Screening of this mass list against an
  appropriate database of peptide fragments
• The fragment database is calculated from a
  protein database by using the known properties of
  the protease
• Usually accurate mass determination of only 5
  tryptic peptides is sufficient for unambiguous
  identification

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Proteolytic digest Cleave of disulfides
SH SH
S S
SH SH
S S
reduction followed by oxidation leads to
scrambling of disulfide bonds
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Thiol group alkylation


Iodoacetamide
Cysteine
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Digestion
AGWNAYIDNLMADGTCQDAAIVGYKDSPSVWAAVPGKTFVNITPAEVGVL
VGKDRSSFYVNGLTLGGQKCSVIRDSLLQDGEFSMDLRTKSTGGAPTFNV
TVTKTDKTLVLLMGKEGVHGGLINKKCYEM ASHLRRSQY
Digest of profilin by trypsin
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Protein Mass Spectrometry
Molecular weight determination Analysis of
digested peptides
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MALDI-TOF-MS

• Mostly singly charged analyte ions (M H),
  minor or no fragmentation
• Mass range of analytes 500-300000 Da
• Resolution with MALDI-TOF-MS ?10.000
• Accurate mass measurements requires internal
  standards
• Mass accuracy
• 500-3000 Da 30 ppm ( 0.1 Da at 3000)
• 3-10 kDa 100 ppm ( 1 Da at 10000)
• 10-30 kDa 300 ppm ( 10 Da at 30000)
• Sensitivity for peptides lt 10 fmol

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ESI TOF MS of enolase showing the many different
charge states of the protein (A) and
deconvolution to the singly charged species (B)
B
A
42000 46000 50000
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Positive-ion ES spectra of FMN-bp recorded under
denaturing conditions
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Peptide sequencing
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Peptide fragmentation
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Peptide fragmentation
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Peptide sequencing
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Peptide sequencing
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De Novo interpretation
A
V
I/L
I/L
I/L
D
D
113
99
71
115
113
113
115
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MS/MS spectrum of the M 2H2 ion of GluFib
y12 y11 y10 y9 y8 y7 y6
y5 y4 y3 y2 y1 Glu-Gly-Val-Asn-Asp-Asn-
Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg b2
b3 b4 b5
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De Novo interpretation
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De Novo interpretation
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MALDI-TOF spectrum of a digest peptide mixture of
a protein
which protein ?
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Protein identification by mass spectrometry and
database searching peptide mass fingerprinting

• Preparation of a proteolytic digest of the
  protein using trypsin
• Determination of mass spectrum of peptide
  mixture experimental mass list
• Screening of this mass list against an
  appropriate database of peptide fragments
• The fragment database is calculated from a
  protein database by using
• the known properties of the protease

1050.5148 1088.4691 1119.4783 1189.6080 1218.6790
1317.7185 1429.7816 1439.8030 1444.6775 1460.6853
1618.8668 1668.8649 1719.8735 1767.9836 1782.8192
1962.9484 2327.1013 2519.2056
dBase
Make list
compare
match
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Bioinformatics peptide mass fingerprinting PMF
MS-Data

• Advanced search engines avalable on the internet
• MASCOT (http//www.matrixscience.com)
• MS-FIT (http//prospector.ucsf.edu)
• PepIndent (http//expasy.ch/tools/peptident)
• Profound (http//65.219.84.5/service/prowl/)
• PeptideSearch (http//www.narrador.embl-heidelber
  g.de)

Protein hitlist Protein A Protein B Protein
C Protein D

• Protein databases available on the internet
• Swissprot / TREMBL
• MSDB (EBI) or nr (NCBI)
• dbEST

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Bioinformatics peptide mass fingerprinting PMF
experimental
in silico
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MASCOT PMF
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MASCOT PMF
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Bioinformatics
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PROBLEM

• Biologist
• What protein was isolated?

• Bank President
• Who robbed the bank?

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GATHER EVIDENCE

• Mass Spectrometrist
• 1. Interview biologist who isolated the protein
• 2. Cleave protein to obtain peptide mixture
• 3. Analyze peptide mixture by MS to obtain
  peptide molecular masses!

• Police Officer
• 1. Interview witnesses
• 2. Dust for fingerprints

enzyme
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DATABASE SEARCH

• Mass Spectrometrist
• Approx. molecular weight 30,000
• Origin bovine liver
• Peptide mass list from MS analysis 975.4832,
  1112.5368, 632.3147, 803.4134, 764.3892

• Police Officer
• Height 57
• Weight 160 lbs
• Gender male
• Age 35-40
• Fingerprints

search
search
DATABASE OF KNOWN FELONS
PEPTIDE MASS DATABASE OF KNOWN PROTEINS
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DATABASE SEARCH RESULTS

• Police Officer
• Identifies the robber
• Mr. Arif Akram

• Mass Spectrometrist
• Identifies the protein
• bovine carbonic anhydrase