Mass Spectrometry Based Proteomics
Proteomics Shared Research
Oregon Health & Science University
Portland, Oregon
This document is designed to give a brief overview of Mass Spectrometry Based Proteomics as we perform it here at OHSU. It covers some of our instruments, techniques, and software and contains:
-A brief introduction to the mass spectrometer
-Description of the protein whole mass determination procedure
-An overview of method used for protein identification including:
-Going from protein to peptides
-MS/MS Fragmentation
-Database Searching
-Guidelines for interpreting results
The Mass Spectrometer
No modern proteomics lab would be
complete without a mass spectrometer. A
mass spec, as it is often called, is a complex
instrument used to obtain the mass of small
molecules including: polymers, proteins,
peptides, and organic compounds, among
other things.
To do this molecules are ionized and then
introduced into an electrical field where they
are sorted by their mass to charge ratio
(m/z). There are many different ways to
ionize the sample, two of the most common
are MALDI (Matrix-assisted laser
desorption/ionization)and electrospray.
In MALDI a laser is used to ionize and
vaporize a small amount of sample, which is
then drawn into the mass spectrometer for
analysis. In PSR we use an atmospheric
MALDI source attached to our
Thermofinnigan LCQ mass spectrometer.
This setup doesn’t have much sensitivity,
but it is very useful for screening samples.
The other common method of ionization is electrospray. Here a stream of liquid containing the sample i
s ionized by applying an electrical charge to it. This creates a stream of ions which repel each other upon exiting the capillary tubing creating a fine plume of ions which is then drawn into the mass spectrometer for analysis.
Types of Mass Analyzers
Once the sample has
been ionized and passed
into the mass
spectrometer it is then
sorted into component
parts by the mass over
charge ratio (m/z).
One common
component used to sort
ions is the quadrupole.
This consists of 2 pairs of
charged rods. There is an
electrical potential
between each pair of rods
which draws the ions
towards one rod. The
polarity of this electrical
field is oscillated rapidly,
which causes the ions
travel through the
quadrupole in a spiral trajectory. Each oscillation frequency allows ions with a particular m/z to pass through, while the other ions crash into the poles and lose their charge, or are
ejected from the quadrupole. By varying the oscillation frequency ions with different m/z ratios will get through. The number of ions passing through at any given frequency is measured by the mass spectrometer’s detector and a graph of intensity vs. m/z is created from this data. This is referred to as an MS Spectra.
Another component used
to filter ions is the ion trap.
In an ion trap ions are
collected and held either in
a 3-dimensional space or a
2-dimentional plane. Once
a certain number of ions
have been collected, or
after a set time, the ions are
ejected from the trap. This
ejection voltage is ramped
in a way that allows
different m/z ions to be
ejected at slightly different
times. This time difference
creates an MS Spectra.
Because a greater number
of ions is collected, this
method typically has a higher sensitivity than using a quadrupole mass filer.
One more common method of sorting ions is the Time-of-
Flight or TOF analyzer. In this analyzer the ions are
collected in a similar manner to an ion trap, and then
accelerated with one push into an empty chamber with an
electrical field in it. The chamber is at a very low pressure,
usually about 1*10-7 torr, this allows the ions to fly freely
with few collisions with other molecules. The ions are
reflected by the electrical field (ion mirror) into a detector.
Because larger m/z ions take longer to be turned around in
the electrical field they arrive at the detector later, allowing
for the creation of a MS Spectra. Because of the way the ions
are sorted this method of analysis has high mass accuracy.
All the mass Spectrometers at PSR employ multiple types
of mass analyzers in their design. These types of mass
spectrometers are sometimes referred to as Hybrid Mass
Spectrometers. For example PSR has no quadrupole only
mass spectrometer; but quads are used to channel/pre-filter
ions in all of our instruments. Here the quadrupole acts as a
‘rough’ filtering device, allowing a subset of the sample
through to be analyzed in greater detail. This type of filtering
reaction massprior to analysis is essential for protein identification, and is used to create tandem mass spectra.
PSR’s Mass Spectrometers
The Proteomics Shared Resource has access to 4 different mass spectrometers. These are the Thermofinnigan LCQ, Thermofinnigan LTQ, Applied Biosystems QSTAR XL, and Applied Biosystems QTRAP 4000.
ThermoFinnigan LTQ
The LTQ quadrupole linear ion trap instrument is the newest
generation of ThermoFinnigan's ion traps and confines trapped ions
in a 2-dimensional space instead of 3-dimensional one.  This allows
confinement of a greater number of ions without degrading
resolution and mass accuracy due to space charging effects,
producing a very sensitive electrospray ionization tandem MS
instrument. The instrument is also capable of scanning at much higher rates than other instruments, typically producing over 15,000 scans during a single LC-MS experiment.  For highest sensitivity the instrument is also fitted with a ThermoFinnigan IonMax nanospray source and packed tip probe.
ThermoFinnigan LCQ
Our LCQ ion trap instrument is fitted with an atmospheric pressure MALDI source from Mass Technologies for rapid collection of MS/MS spectra from unfractionated protein digests.  This automated system can identify approximately 100 proteins in 4 hours of analysis time, and is especially useful for samples produced from gel digests where sample complexity is not great.  While this platform has a lower sensitivity it is more cost effective since it requires far less instrument time than LC-MS analysis. Applied Biosystems QSTAR XL
This hybrid quadrupole-TOF mass spectrometer is equipped
with both PicoView (New Objective) PV400 nanosources or
IonSprayer sources (MDS Sciex) for sample introduction.  The
ability to collect high resolution, high mass accuracy (better
than 20 ppm) MS and MS/MS data with this instrument is a
key capability in performing protein identification, expression
analysis, and analysis of post-translational modifications.  The
mass spectrometer is capable of resolving ions from 50-6000
m/z in resolving mode and 45- 40,000 m/z in transmission only mode. Resolution in resolving mode is generally better than 8,000 FWHM. A unique pulsing function enables sensitive precursor ion scanning on this instrument which is valuable in the analysis of
post-translational modifications. Because of its high mass accuracy the QSTAR is typically used by PSR for whole protein mass determination experiments.
Applied Biosystems QTRAP 4000
Multiple reaction monitoring (MRM) experiments are
carried out with this instrument which contains, an
advanced hybrid triple quadrupole/linear ion trap.  This
instrument uses MDS SCIEX Linear Ion Trap (LIT)
technology, which enables excellent quantitative and
qualitative performance within a single system. Use of a
quadrupole as a linear ion trap significantly enhances ion trap performance by increasing ion capacity, improving injection and trapping efficiencies, and increasing duty cycle.  This system provides true triple quadrupole MRM at the highest level of sensitivity, as well as extended dynamic range, ensuring superior quantitation performance for peptides.  The Q TRAP can be configured with either the TurboIonSpray source or the MicroIon source, mounted on the MDS Sciex nanosource.
The unique design of the QTRAP 4000 allows for alternate quadrupoles to allow different charged particles (+/-) through. For example a positive ion can be let through the 1st quadrupole, be broken ap
art in the 2nd quadrupole, and it’s negatively charged components can be passed through the final quadrupole for analysis. This unique ability makes the QSTAR 4000 especially good at identifying phosphorylation sites as the phosphate group is negatively charged and peptides carry a net positive charge. Whole Mass Determination
One of the services offered by the Proteomics Shared Resource is the determination of the mass of a whole protein. This can be useful of determining which form of a protein is present, whether or not a substrate has bound to a protein, or for characterization of a protein complex.
In this method sample is
infused into the mass
spectrometer and a series of MS
spectra are created. These spectra
can be summed over a period of
time, up to 15 minutes, allowing
for the detection of even small
amounts of protein. When the
composite MS spectra is viewed
a series of peaks are seen. These
peaks represent the different
charge states of the protein.
A computer program is used to
identify all the different charge

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