Fragmentation of Protonated Peptide Ions via Interaction

Fragmentation of
Protonated Peptide Ions
Group meeting
Thursday, August 31, 2006
Introduction on proteomics
Use of mass spectrometry and different MS-MS techniques:
•Fragmentation patterns
Details of an ETD MS-MS published recently
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
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
Protein diversity can not be fully characterized by gene expression
analysis alone, making proteomics a useful tool for characterizing cells and
tissues of interest.
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
Collision Induced Dissocation
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
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
Peptide structure
Fragmentation nomenclature
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)
Fragmentation of Protonated
Peptide Ions via
Interaction with Metastable
Vadym D. Berkout
MassTech, Inc., Columbia, Maryland
Anal. Chem.2006, 78,3055-3061
Electron Transfer Dissociation
A negative or neutral atom will transfer the electron to peptide for fragmentation
M n+ + Ar*
M (n-1)+ + Ar +
Glow discharge a source for metastable argon
25 Torr
5 mTorr
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.
Octopole Ion guide
Quadrupole Ion guide
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
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
Fragmentation of peptides in trapping mode
Peptide ions produced by electrospray were trapped in quadrupole for 100-400 ms
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
Substance P
Lots of c fragments
Similar to ECD
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.
complete c and z fragments
Except c1 (same as ECD)
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
Fibrinopeptide A
Lots of b and y fragments in
addition to c and z fragments
Potential energy diagram for interaction of peptide cation
with metastable electronically excited atom.
Dependence of cross section of the electron transfer
on the collision energy.
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.
Other refrences: