LC-MS/MS
general information
--- LC-MS/MS
is a technique that combines the solute separation power of
HPLC, with the exquisite detection power of a mass spectrometer.
HPLC can separate peptides on the basis of a number of unique
or species specific properties of peptides such as charge,
size, hydrophobicity and presence of a specific tag or amino
acid(s). HPLC is also an excellent way remove potentially
interfering molecules from the sample such as salts, buffers
and detergents. These types of molecules greatly influence
the efficiency of the ionization and the quality (and quantity)
of data generated by the MS is greatly dependent on a clean
sample prior to ionization. Coupling a high performance liquid
chromatography (HPLC) system with a mass spectrometer has
proved to be a difficult task and a great deal of research
has gone into this problem. The difficulty has been that the
HPLC system deals with analyte in the liquid-phase yet the
MS requires a transformation of these ions from the liquid
phase to ions in the gas. It is challenging to maintain a
sufficient vacuum level in the mass spectrometer because introduction
of a liquid at the ion source wreaks havoc on the vacuum.
For this reason the solvent must be stripped and gas phase
ions must be generated before introduction to the MS. LC/MS
for practical use with biopolymers such as proteins made leaps
and bounds with the introduction of the “thermospray”
interface. The next big improvement was the introduction of
the electrospray and APCI techniques which are both API (atmospheric
pressure ionization) techniques. These methods allow ionization
at atmospheric pressure and both are considered to be a method
of soft ionization which is a major prerequisite to the analysis
of proteins. Because the HPLC is coupled to a tandem mass
spectrometer with an ESI interface we can rapidly separate
complex protein mixtures and identify its components. We will
discuss each component of this system in more detail below,
namely
--- 1)
Why LC-MS/MS and what can it do for our clients?
--- 2) HPLC (high performance
liquid chromatography).
--- 3) ESI ionization (electrospray).
--- 4) Tandem (Quadrupole - Time
of Flight) mass spectrometry.
The latter two topics (3 and 4) will be discussed
very briefly since a basic mass spectrometry tutorial is available
in the “resource” section of our website that
explains these aspects in more detail.
1) Why LC-MS/MS and what can it do
for our clients?
--- LC-MS/MS
is used for identification of proteins and is the method of
choice under a number of circumstances. Often MALDI-ToF MS
experiments are run on samples such as in-gel or in-solution
digests from purified protein sources. MALDI-ToF is a more
general technique and when used for protein identification
is often limited to pure proteins or at least very simple
mixtures. This is due to the fact that this method relies
on measurements of multiple peptides generated from digests
of a parent protein. These peptide masses or “the peptide
mass fingerprint for a given protein” (a list of masses)
are then searched against sets of masses from a database of
theoretically digested proteins. If peptides from multiple
species are present in this mass list it becomes extremely
difficult to distinguish which peptide (m/z) belongs to which
protein and the search algorithm is likely to fail. So where
are we going with this? Well, this same sample that caused
problems on the MALDI-ToF instrument is a prime candidate
for generating excellent results by LC-MS/MS for a number
of reasons. First, LC-MS/MS will also produce a spectra in
much the same as the mass fingerprint generated by the MALDI-ToF
but in addition, MS/MS experiments will generate peptide primary
structure (sequence) information from this peptide. This is
an additional level and very specific type of information
that has a greater chance of producing a positive identification
from a heterologous database. Advantages:
This is the most cost-effective method for generating primary
sequence information (identifications) from proteins. It is
easily automated and therefore makes high-throughput anaysis
possible. Reverse phase separation also effectively concentrates
peptides and elutes peptides of the same species at the same
time. Therefore the same sample can appear to generate a more
intense signal than it did by manual nanospray MS/MS or by
MALDI-ToF anaysis. This method can also deal with relatively
complex mixtures. Disadvantages: If the protein(s)
of interest is(are) not in the database or if no protein exists
with sufficient homology then identification can be problematic.
De novo sequencing is possible to permit BLAST searching with
sequence data that is interpreted from individual spectra.
Has an upper limit to the managable complexity of the sample,
two-dimensional (or even more) levels of separation are needed
for very complex mixtures.
2) High-performance liquid chromatography
(LC or HPLC)
--- We will
discuss HPLC in its most basic form (two solvents with dual
pumps and a linear gradient) and its use with a reverse-phase
(RP) column. Reverse-phase columns separate peptides on the
basis of hydrophobicity. Reverse-phase is the workhorse column
in most single dimension LC-MS/MS experiments. When multiple
dimensions of liquid separation are required (complex protein/peptide
mixtures) additional columns are used with properties as orthogonal
as possible from reverse-phase, such as charge (anion or cation
exchange). Additional columns will be discussed in the 2D-LC-MS/MS
section in the “science” section of our website.
Compounds stick to reverse phase HPLC columns in high aqueous
mobile phase and are eluted from RP HPLC columns with high
organic mobile phase. Peptides would rather stick to the “greasy”
hydrophobic column material than be solubilized in the 100%
water (aqueous) phase. Peptides can be separated by running
a linear gradient of the organic solvent. As the organic solvent
increases, species of peptide begin to become solubilized
in the gradually increasing organic phase. The more hydrophobic
the peptide the higher the organic concentration must be before
the peptide will come off of the RP column. In this type of
experiment two separate buffers are needed and therefore the
HPLC instrument must be able to pump and control the concentration
of two buffers independently.
HPLC Columns-
HPLC column dimensions are defined by “internal diameter
x length” (4mm X 200mm). Many columns are packed with
silica beads that are defined by their particle (bead) and
pore (molecular sieve) size. Particle sizes can vary from
3 and 50 um and a majority of peptide work is done with 5
um beads. Smaller particles usually offer higher separation
(resolution of different peptide species) but will also generate
more pressure than larger beads. The pore size is measured
in angstroms (A) with a popular RP column pore size for peptides
being about 300 A. Although silica is a good inert support
for packing columns, it will break-down at higher pH and therefore
is used under acidic conditions. If conditions must be at
pH above 7.0 then a different bead must be used to pack the
column. Generally all RP chromatography is carried out at
low pH. This is not only to protect the silica but also the
chemistry of the peptides at low pH is favourable for RP separation
and ionization for MS analysis. The stationary phase is made
up of hydrophobic alkyl chains -CH2 (methyl groups) of which
there are three common alkyl chain lengths, C4 for proteins,
C8 for proteins and peptides and C18 for peptides and other
small molecules. Peptides would not stick well to a C4 column
and on the other hand a whole intact protein may never come
off of a C18 column.
Solvents and gradients-
The reverse phase solvents are normally installed on
the HPLC channels A and B. Unless you are from Mars or have
a broken channel on your LC system the A solvent is the aqueous
buffer (acidified water) and solvent B is the organic solvent
(acidified acetonitrile). The A solvent is generally HPLC
grade water (often with a very small organic component of
2-5% with 0.1% formic acid. The B solvent is generally an
HPLC grade organic solvent such as acetonitrile with 0.1%
to 0.05% formic acid. The acid is used for a couple of reasons.
The low pH causes all the peptides to have an overall positive
charge and the acid ion acts as an ion-pairing reagent to
"hide" that charge. This optimizes the separation
since it is based soley on hydrophobicity and repelling charge
effects should not play a role in sepration. The acid is also
compatible with silica RP beads and serves as a source of
protons in reverse phase LC/MS that are needed for fragmentation
and for a form of collisionally induced dissociation (CID)
involving a mobile proton.. For uncharacterized sample a basic
starting gradient is 0% B to 60% B in 60 minutes. That is
0% percent organic mobile phase at time zero and gradually
increases by 1% organic (acetonitrile) every minute. After
sixty minutes 60% organic content is reached and almost all
species of peptides should be well off the column. The column
needs to be re-equillibrated at 0% B once again before use
or the next time peptides are loaded onto this column they
would "slip" right through? This highlights the
importance of knowing the organic component of any sample
as it could be lost during various types of chromatography
due yo buffer incompatability or other solute interference.
3) ESI ionization (electrospray).
coming soon - please see the mass spec tutorial
in the resources section also coming soon
4) Tandem (Quadrupole - Time of Flight) mass spectrometry.
coming soon - please see the mass spec tutorial in the resources
section also coming soon
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