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Biological Mass Spectrometry
A mini course
Biological Mass SpectrometryGeneral Mass Spectrometry
The General mass spectrometerAnalyzers typesCalibrations and mass AccuracyDetection
Matrix Assisted Laser Desorption (MALDI) TOF
Electrospray Ionization (ESI)Quadrupoles, Ion Traps, FTMS, and O-TOF
FragmentationPost source dissociation (PSD) (with MALDI)In source dissociation (with ESI)Collision induced dissociation CIDIRMPD and ESI
The General Mass Spectrometry
Ion S ou rceM A L D I
S p ray S ou rces
A n a lyzerTO F
Q u ad ru p o lesTrap p in g in s tru m en ts
D etec to r
M ass S p ec trom eter
Analyzers
• Time of Flights mass spectrometer
• Quadrupole mass spectrometer
• Quadrupole Ion traps
• FT-MS, FT-ICR
Time-of-flight Mass Spectrometers
• Basically a tube that ions travel through with lower mass ions traveling quicker through the tube.
• Reflectron increases resolution by compensating for energy and increasing path length.
• Delayed extraction increases resolution.
O-TOF
Ion BeamPulser
Grid
To TOF
Quadrupole Mass Spectrometer
+
+
-- ++
-
-
Rods
Ions
Quadrupole Ion Traps
+
+
-- ++
-
-
Ions
End Caps
Ring
FT-MS, FT-ICR
B+
+
Calibration
• All Mass Spectrometers MUST BE CALIBRATED. This adjust parameters in the mass assignment of the ions.
• In general, the closer in space and time the calibration is the more accurate the calibration.
• Default calibrations are usually set up on instruments for nominal mass accuracy.
• Calibrations can be done with any compound with a known elemental formula. The best calibration compounds are similar type of compounds to the type you are going to measure accurately.
• It is best to have calibration compounds above and below the compound of interest
Mass Accuracy
• Higher resolution allows for higher mass accuracy in two ways– Resolves stardards and sample from background– Better centroiding of peaks
• Use of internal standards with a high resolution (>10,000) is the “Gold standard” for mass accuracy.
• Mass Accuracy needed is dependent on use and available information.
R Voltage across resistor
Ion Detection
WHY MALDI?
• Less Sensitive to Salts• Lower PRACTICAL detection limits• Easier to interpret spectra (less multiple
charges)• Quick and easy• Higher mass detection • Higher Throughput (>1000 samples per
hour)
Matrix
• Needs to be involatile (most are solids at room temperature)
• Needs to absorb the laser wavelength that you are using. (most cases 337 nm)
• Preferably dissolves in same solvent as the sample
• Typically, the matrices are acidic.
HOT and Cold Matrices
• DHB is a cold matrix, the samples are not as likely to be fragmented, may not ionize some molecules.
• Alpha-cyano dihydroxybenzoic acid is considered a hot matrix. More likely to fragment the molecules. Can produce multiply charged proteins.
Sample preparation
• Dried Droplet- mix sample with matrix and drop on plate at the same time. –Easy to do.
• Layered method- put matrix on plate then dry before adding sample. Good for low concentrations, more difficult.
• Want high Matrix-Analyte ratio.
The MALDI Instrument
S ou rceD e layed E xtrac tion
A n a lyzerR eflec tron
P S D
D etec to rW h y d e flec t th e ion s?
Tim e-o f F lig h tM ass S p ec trom ete r
Basic Physics
• Kinetic Energy= ½ mv2
• Potential Energy of charged particle=qV
• Potential Energy +Kinetic Energy=Constant
Delayed Extraction
• Kinetic Energy of ions leaving the surface is not constant. Also, their could be a time delay, in addition.
• Delayed Extraction is a method used to compensate for the spread of kinetic energy of the ions leaving the surface.
• Delayed Extraction is mass dependent.• Delayed Extraction will also compensate for some
time delay of ions coming off the surface.
M1 kE < M2 kE
Pote
ntia
l Volta
ge
Sam
ple
pla
te
M1M2
T=t4
E=kE +PEM1 kE< M2 kE
M1 kE > M2 kE
Dete
cto
r
Reflectron
• The reflectron is another method used to compensate for the spread in Kinetic energies in the time of flight.
• Reflectron does not compensate for any time delay coming off the surface.
• Three types of reflectrons ( single stage, two stage, and curved field)
M1M2
KE M1 > KE M2
M1
M2
KE M1 > KE M2T=t2
M1
M2KE M1 > KE M2
T=t3
KE M2=KE M1=0
T=5
Why Deflect the ions?
• Detector is made up of multiple detectors similar to the electron multiplier called “channels”.
• When an ion strikes the detector the “channel” is dead for milliseconds.
• When to many matrix ions hit the detector, it kills the signal.
• Linear detector is specially design to compensate for larger number of ions, but is not perfect.
Why Electrospray Ionization?
• Electrospray Ionization can be easily interfaced to LC.
• Absolute signals from Electrospray are more easily reproduced, therefore, better quantitation.
• Mass Accuracy is considered better.• Multiple charging is more common then
MALDI.
Detection of Peptides
MALDIDetectedPeptides
Advantages of Multiple Charging
• Can use instruments with lower maximum m/z (i.e., Quadrupoles, ion traps, FTMS)
• For FTMS, the resolution is better at lower m/z values, therefore, ESI helps one obtain better resolution at higher m/z values.
• Multiply charge ions tend to fragment easier then singly charge ions.
To Pumping
- 3kV
Basic ESI Source
Needle options
• High flow needles (for connection to regular LC columns(up to 1ml/min))
• Nanospray needles (high sensitivity work)• Microspray needles (connected to micro LC
columns)• Atmospheric Pressure Chemical Ionization
(for non-polar molecules)• Multiple needles setups
Why Fragment the ions?
• Molecular weight is only one piece of data the instrument can provide
• Data base searches on tryptic peptides do not always reveal the protein.
• Sequence and modification information can be obtained by fragmentation
Ways to Induce Fragmentation
• Post Source Decay-MALDI
• In source Dissociation-ESI
• Low Energy –High Energy Collision induced dissociation
• Inferred multi-photon dissociation (in a trap)
• Electron Capture dissociation (in a trap)
Tandem Mass Spectrometers
• Traps
• Methods using single MS’s
• Triple Quadrupoles
• Q-Tofs
• TOF-TOF
PSD
• Easily done on simple MALDI with reflectron
• Might not produce enough fragments
• Relatively low precursor ion selection
Physics Reminder
• Conservation of Energy assures that the kinetic energy of fragments is determined by the relative masses of the products.
• KE=1/2mv2
• KEprecursor=KEproduct ion+KEproduct neutral
• vprecursor=vproduct ion
• KEproduct ion=KEprecursor*(mproduct ion/mprecursor)
M > M 2> M 2
K > 2> K 21
M > M 2> M 2
K > 2> K 21
M > M 2> M 2
K > 2> K 21
Velocity in Field FreeRegion on the samefor both fragments
Types of Reflectrons• Two stage- Give best resolution on single MS
mode, requires doing PSD in Stages.• Single Stage-Large Reflector takes a significant
amount of space. PSD can be done in single stage, however, low mass product ions have poor resolution.
• Curved field-Another Large Reflector that takes up space. Allows PSD to be obtained in single stage with good resolution throughout PSD, but single MS mode does not have as good a resolution.
In Source Dissociation
• This is done by increasing the voltage between the entrance to the sample orifice and the first skimmer.
• Can be done on simple electrospray instrument.• Produces results similar to low energy CID• No mass selection, therefore, requires sample to
be extremely pure.• Can be used to produce multiple MS on triple and
hybrid instruments.
CID
• CID is divided into low energy (<100 eV) and high energy(>1000 eV) based on the collision energy.
• High energy CID produces more fragments, but is more complicated to interpret.
• Low energy CID has a limit on the m/z it can dissociate of approximately 1000.
• High energy CID produces charge remote fragmentation that can be used to distinguish leucine and isoleucine.
IRMPD and ECD
• Complimentary techniques
• Can only be performed in trapping instruments-Mostly FTMS.
• ECD requires a multiply charged ion
• Used for dissociating high molecular weight fragments
Interpretation of Tandem Mass Spectra of Peptides
• Known Sequence- Calculate expected fragments and compare to tandem spectra to see match
• Modified Sequence-Calculate unmodified sequence compare to tandem spectra to see difference where modification occurs.
• Unknown Sequence- Check Database to see if it is a match
• Unknown Sequence not in Database- Manual Interpretation (Practice!Practice!Practice!)
Peptide fragmentation
-RCH-C-NH-CHR-O
x y z
cba
Nterm Cterm
Manual Interpretation
• Goal-Assign as many abundant fragments as possible to a spectra
• Remember Cysteine modifications• Know type of fragments that are typically
observed by dissociation method.– Low Energy (b and y, loss of neutrals from these
fragments)– High Energy (x,y,z, a,b,c, v, d,w)Reference: Ioannis A Papayannopoulos, Mass Spec.
Rev.,1995, vol. 14, 49-73.
Steps in Manual Interpretation• Choose a large peak• Look for ions that are different in mass from the
fragment by a specific amino acid mass. If there is look for an other fragment that differs by a specific amino acid mass to build a partial sequence.
• Decide the type of fragments that corresponds to the above sequence by either mass difference to the precursor or complimentary fragments.
• Look for other complimentary fragments and low mass ions to confirm the partial sequence.
• Choose another unassigned peak and repeat procedures until sequence is determined.
Peptide modifications to helpInterpretation
• O18 water digestion- all C terminal fragments will have unique Isotope pattern
• N-terminal or C-terminal modifications-results in distinct fragmentation patterns.
LC Interfaces
• Direct LC with Electrospray.– Direct connection to mass spectrometer– Real Time monitoring
• Vacuum deposition for MALDI– Indirect conection to mass spectrometer– Not Real Time, allows one to make decisions
later. Multiple MS/MS on single LC peaks.
LC detection methods
• Simple acquiring of spectra.
• Precursor and product ion scans
• Data Dependent scans
• Selective Ion Monitoring
• Selective Reaction monitoring
Applications
• Protein Identifications
• Protein Modifications
• Bacteria Identifications
• SNP’s
Protein Identification
• Identify Protein mass• Digest Protein• Analyze by MALDI• Data Base Search• If negative results, go to Tandem Mass
Spectrometry• Data Base Search on Tandem Mass
Spectrum
Protein Modifications
• Tandem Mass Spectrometry is very important• Can obtain full mass of proteins to help determine
mass and number of modifications• Digest proteins and run mass spectrometry.• Use Tandem to determine the exact position• Use of unmodified protein or peptide can be useful
in the interpretation
Bacteria Identification
• Uses MALDI of Bacteria to Identify Proteins produced by bacteria
• Data base search of MALDI spectra identifies bacteria
• At this point, no large consistent Data Base is available.