Fragmentation of Protonated Peptide Ions

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Fragmentation of Protonated Peptide Ions

• Group meeting
• Thursday, August 31, 2006

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Outline

• Introduction on proteomics
• Use of mass spectrometry and different MS-MS
  techniques
• CID
• ECD
• Fragmentation patterns
• Details of an ETD MS-MS published recently

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Introduction
Proteomics The large-scale study of proteins,
particularly their structures and functions.
The entirety of proteins in existence in an
organism throughout its life cycle are referred
to as the proteome of the organism. Protein
identification. Is one of the important branches
of proteomics. Well-known methods are 1-Edman
degradation low-throughput sequencing, limited
to peptides of 50-60 amino acids 2-Mass
spectrometry Higher-throughput, can identify
much larger peptides and even proteins
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Proteomics vs. genomics
In the human genome there are 22,000 genes vs.
400,000 proteins. This makes proteomics more
complex than genomics. The large increase in
protein diversity is thought to be due to
alternative splicing (mRNA change) and
post-translational modification of proteins.
Protein diversity can not be fully
characterized by gene expression analysis alone,
making proteomics a useful tool for
characterizing cells and tissues of interest.
http//www.scientificpsychic.com
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Mass spectrometry in proteomics

• Protein or peptide is identified from its
  molecular weight and the mass to charge value of
  its fragments
• Tandem mass spectrometry (MS-MS) is one of the
  most important methods for protein and peptide
  identification
• High sensitivity (femtomole level, 10-15) and
  specificity
• Collision induced dissociation (CID) of
  protonated peptide cations is the most common
  method for fragmentation
• Electron capture dissociation (ECD) is also used
  as a complement of CID

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Collision Induced Dissocation (CID)
Multiple collisions of gas molecules with
peptide. The kinetic energy of neutral gas
molecule transfers to the peptide, it will
rapidly distribute all over the covalent bonds in
the molecule and this will lead to amide bond
breakage. Cleavage of the peptide amide bond to
produce b and y-type fragment ions. Incomplete
backbone fragmentation and loss of labile groups
that are very important in posttranslational
modifications
Q3
Q1
Q2
http//www.city.sapporo.jp
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Peptide structure
Ca
http//courses.cm.utexas.edu/emarcotte/ch339k/fall
2005/Lecture-Ch3-1/Slide18.JPG
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Fragmentation nomenclature
http//www.matrixscience.com/help/fragmentation_he
lp.html
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Electron Capture Dissociation (ECD )
Capture of a thermal electron by a protonated
peptide causes the peptide fragmentation from
N-Ca bond. This will produce N-terminal
c-fragments and C-terminal z-type fragment
ions. Unlike CID this is nonergodic, meaning
that the fragmentation happens before the energy
transfer within the molecule. As a result labile
modification groups will remain intact. The
maximum cross section is for thermal electrons
Only successful in FT-ICR mass spectrometry.
(strong RF electric field of quadruple and ion
trap makes the introduction of such electrons
difficult)
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Fragmentation of Protonated Peptide Ions
via Interaction with Metastable Atoms
Vadym D. Berkout MassTech, Inc., Columbia,
Maryland
Anal. Chem.2006, 78,3055-3061
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Electron Transfer Dissociation (ETD) A negative
or neutral atom will transfer the electron to
peptide for fragmentation M n Ar
M (n-1) Ar
Fragmentation
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Glow discharge a source for metastable argon
25 Torr
5 mTorr
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GD source Asymmetric electric field better
separation of metastable atoms from
ions. Cathode 1.5 mm diameter, oxygen free
copper rod Anode off-axis planer Voltage 300V,
I 5mA Two lenses plus a nickel grid are
charged with few hundred volts negative potential
to prevent any electron entrance into the
quadrupole region.
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Octopole Ion guide
Quadrupole Ion guide
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Only metastable argon?
M Ar . M . Ar
charge exchange ionization (15.7 eV
1520kJ/mol) M Ar M .
Ar e Penning ionization (11.72 eV and
11.55 eV)
Ionization energy for different gases (eV)
Nitrogen 15.58 Methane 12.61 Ammonia
10.07 Isobutene 10.68
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15.58
12.61
10.07
10.68
Argon is mainly neutral and the small amount of
argon ions will be ejected from quadrupole by
setting the low mass cut off at m / z 250
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Fragmentation of peptides in trapping mode
Peptide ions produced by electrospray were
trapped in quadrupole for 100-400 ms
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Substance P 11-amino acid polypeptide with
the sequence Arg Pro Lys Pro Gln Gln Phe Phe
Gly Leu Met NH2. In central nervous system The
regulation of mood disorders, anxiety, stress,
reinforcement, neurogenesis, respiratory rhythm,
neurotoxicity, nausea / emesis and pain
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Substance P
Lots of c fragments Similar to ECD
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Bradykinin Small peptide with 9 amino
acids. arg - pro - pro - gly - phe - ser - pro -
phe - arg Bradykinin is a potent
endothelium-dependent vasodilator, causes
contraction of non-vascular smooth muscle,
increases vascular permeability and also is
involved in the mechanism of pain. In some
aspects, it has similar actions to that of
histamine, and like histamine is released from
venules rather than arterioles.
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Bradykinin
complete c and z fragments Except c1 (same as ECD)
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Fibrinopeptide A A peptide released as part of
the clotting process. Mw 1536.6
Thrombin clips two peptides (fibrinopeptide A and B )from fibrinogen. This produces the fibrin monomer with exposed polymerization sites that can bind to other fibrin monomers. The monomers polymerize to form a loose clot
http//www.hematology.org
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Fibrinopeptide A
Lots of b and y fragments in addition to c and z
fragments
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Potential energy diagram for interaction of
peptide cation with metastable electronically
excited atom.
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Dependence of cross section of the electron
transfer on the collision energy.
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It shows that metastable argon atoms can be a
source of electron for peptide dissociation.
This allows ETD in Ion trap and quadrupole as a
substitute of ECD in FTMS.
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Other refrences http//www.answers.com http//en
.wikipedia.org http//www.mcb.harvard.edu/DonWiley