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Proteomics and Mass Spectrometry Core
Laboratory
Mass Spectrometry
Theory
Mass Spectrometry
Mass spectrometry measures the mass of
molecules by measuring the mass-to-charge ratio (m/z). Mass is a
molecular attribute that can help identify or confirm the identity of a
molecule.
Molecular weight measurements by mass spectrometry are based upon the production, separation, and detection of molecular ions. A typical mass spectrometer includes:
- Ion source—Ionizes sample and generates gas phase ions
- Analyzer—Separates ions according to individual mass-to-charge ratios
- Detector—Detects and amplifies ions
- Data system—Converts detection of ions into a readable or graphic display
MALDI-Process
In MALDI the sample is co-crystallized
with an excess of an UV-adsorbing matrix, a low molecular weight compound
that enhances the de-adsorption and ionization of intact sample molecules.
The so formed ions are then extracted by applying an electric field
Often used matrices:
-
ACCA:
α-cyano-5-hydroxy
cinnamic acid
-
DHB:
2,5-dihydroxy benzoic acid
-
DHBs: (9+1)
2,5-dihydroxy benzoic acid/2-hydroxy-5-methoxy benzoic acid
- HPA: 3-hydroxy picolinic acid
- THAP: 2,4,6-trihydroxy acetophenone
TOF
Time-of-flight mass spectrometry works on the principle that if ions are
accelerated with the same potential from a fixed point and at a fixed
initial time and are allowed to drift, the ions will separate according to
their mass-to-charge ratios. Lighter ions drift more quickly to the
detector. Heavier ions drift more slowly. Doubly and higher charged ions
drift faster than singly charged ions of the same species.
With: Ekin = 1/2 m v2= zU
-> v=[(2zU)/m]1/2 and v = s/t
-> t = [s/(2U)1/2]*(m / z)1/2
were: m = mass
v = velocity
t = drift time -> -> m/z = const*t2
s = drift distance
z = charges per ion
Ekin = kinetic energy
U = acceleration voltage
Instruments
Voyager-DE STR
(Applied Biosystems, Foster City, CA, USA )
The Voyager-DE™ STR Biospectrometry™
Workstation is a floor-standing MALDI-TOF (matrix-assisted laser desorption
ionization time-of-flight) mass spectrometer that includes a reflector
analyzer.
The features of this instrument include positive and negative ion detection, linear or reflector mode operation and post-source decay (PSD) analysis capability.
Extremely usefull in the lower mass range of 1000 to 5000 Da, (sensitivity in the low femto mole range with dried droplet application, mass accuracy +/- 0.01 Da and better) needed e.g. for peptide fingerprinting of proteins, the theoretical m/z range exceeds 300 kDa.
Voyager instrument schematics:
QStar Pulsar i
The
QStar Pulsar I is a hybrid quadrapole time-of-flght mass spectrometer. This
instrument is state of the art for high throughput sequencing of
biopolymers. It is equipped with both nano electrospray ionization (ESI) and
orthogonal matrix assisted laser desorption/ionization (oMALDI) ion sources.
The QStar Pulsar I allows for the following ultra sensitive analyses:
- Accurate oMALDI mapping of peptides, oligosaccharides and glycoconjugates
- oMALDI sequencing of biopolymers with Mr<3 000
- nanoESI sequencing of biopolymers with m/z<=3000
- High-throughput peptide sequencing
- Phosphopeptide analyses
- O-glycopeptide analyses
- N-glycopeptide analyses (limited to nanoESI for N-glycans with Mr>3000)
- Precursor ion scanning experiments
- Information dependent acquisition experiments
- nanoESI accurate protein MW determination
- nanoESI protein-protein, protein-ligand and other non-covalent complexes measurements
Ciphergen Protein Chip Reader
MSCF has also installed
the Ciphergen SELDI-TOF (Surface Enhanced Laser Desorption/Ionization
Time-Of-Flight) mass spectrometer. This instrument is optimized for
measurement of proteins, rather than peptides. The chips’ purpose is
covalent and non-covalent binding/profiling of proteins of interest in order
to enhance sensitivity, enable work with raw biological material and to
enable advanced biological interaction/MS studies.
The special-features of chip surfaces can be divided in 3 categories:
- Chromatography surfaces: NP (normal phase), WCX (weak cation exchange), SAX(strong anion exchange) chips
- Metal affinity surface: IMAC3 (immobilized metal affinity chip) chip for metal binding, which can subsequently be used for capture of phosphopeptides, histidine containing peptides, or acidic peptides.
- Preactivated surfaces: PS1 (contains a carbonyldiimidazole surface which covalently reacts with amine groups) and PS2 (contains an epoxy surface which covalently reacts with amine and thiol groups) chips. DNA and proteins, including antibodies, can be immobilized on the PS1 and PS2 surfaces.