fragmentation of protonated peptide ions group meeting thursday, august 31, 2006
TRANSCRIPT
![Page 1: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/1.jpg)
Fragmentation of Protonated Peptide Ions
Group meeting
Thursday, August 31, 2006
![Page 2: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/2.jpg)
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
![Page 3: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/3.jpg)
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
Introduction
![Page 4: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/4.jpg)
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.
Proteomics vs. genomics
http://www.scientificpsychic.com
![Page 5: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/5.jpg)
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
Mass spectrometry in proteomics
![Page 6: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/6.jpg)
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
Collision Induced Dissocation (CID)
Q1 Q2 Q3
http://www.city.sapporo.jp
![Page 7: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/7.jpg)
http://courses.cm.utexas.edu/emarcotte/ch339k/fall2005/Lecture-Ch3-1/Slide18.JPG
Peptide structure
C
![Page 8: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/8.jpg)
http://www.matrixscience.com/help/fragmentation_help.html
Fragmentation nomenclature
![Page 9: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/9.jpg)
Capture of a thermal electron by a protonated peptide causes the peptide fragmentation from N-C 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)
Electron Capture Dissociation(ECD )
![Page 10: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/10.jpg)
Vadym D. Berkout
MassTech, Inc., Columbia, Maryland
Anal. Chem.2006, 78,3055-3061
Fragmentation of Protonated Peptide Ions via
Interaction with Metastable Atoms
![Page 11: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/11.jpg)
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
![Page 12: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/12.jpg)
5 mTorr
25 Torr
Glow discharge a source for metastable argon
![Page 13: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/13.jpg)
GD source
Asymmetric electric field: better separation of metastable atoms from ions.
Cathode: 1.5 mm diameter, oxygen free copper rodAnode: off-axis planerVoltage = 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.
![Page 14: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/14.jpg)
Octopole Ion guide
Quadrupole Ion guide
![Page 15: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/15.jpg)
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: 15.58Methane: 12.61Ammonia: 10.07Isobutene: 10.68
Only metastable argon?
![Page 16: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/16.jpg)
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
15.58 12.61
10.07 10.68
![Page 17: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/17.jpg)
Fragmentation of peptides in trapping mode
Peptide ions produced by electrospray were trapped in quadrupole for 100-400 ms
![Page 18: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/18.jpg)
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
![Page 19: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/19.jpg)
Substance P
Lots of c fragmentsSimilar to ECD
![Page 20: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/20.jpg)
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.
![Page 21: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/21.jpg)
Bradykinin
complete c and z fragmentsExcept c1 (same as ECD)
![Page 22: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/22.jpg)
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
![Page 23: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/23.jpg)
Fibrinopeptide A
Lots of b and y fragments in addition to c and z fragments
![Page 24: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/24.jpg)
Potential energy diagram for interaction of peptide cationwith metastable electronically excited atom.
![Page 25: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/25.jpg)
Dependence of cross section of the electron transferon the collision energy.
![Page 26: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/26.jpg)
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.
![Page 27: Fragmentation of Protonated Peptide Ions Group meeting Thursday, August 31, 2006](https://reader035.vdocuments.site/reader035/viewer/2022062304/56649e3c5503460f94b2e588/html5/thumbnails/27.jpg)
Other refrences:
http://www.answers.comhttp://en.wikipedia.orghttp://www.mcb.harvard.edu/DonWiley