Download Sample Preparation for Peptides/Proteins

Sample Preparation for Peptides/Proteins
If your sample is a complete unknown (you have no idea what the mass is) you may
want to start with SA. Usually all peptides/proteins respond with SA. Then you can
optimize the conditions depending on the peaks you observe in the spectrum. The
general rule for MALDI analysis for this class of samples is based on their mass.
If you have an analyte with a mass below 10000 Da, CHCA is the matrix of choice. For
larger proteins (> 10000 DA), SA works best. A simple protocol for sample preparation
follows (for a detailed graphical presentation of the sample procedure read: "MALDI in
the Lab"):
1) Adjust the concentration of your sample. The optimum concentration for
peptides/proteins is between 5-50 pmole/L (this will give a final concentration of 0.5-5
pmole/ after mixing with the matrix). If the concentration is unknown make a series
of dilutions in 0.1%TFA.
2) Prepare a saturated solution of the matrix in a 1:1 v/v ACN/0.1%TFA solution.
Vortex the mixture for 60 seconds and then centrifuge it for 20 seconds. Use only the
supernatant solution. This is to prevent any inhomogeniety in the crystallization, since
any particulate in the solution can act as a nucleation site and cause inhomogeniety in
the crystallization.
3) Transfer 9  of the matrix to a 0.5 ml eppendorf tube. Add 1  of the analyte and
either mix with the tip of your pipette tip or vortex for 5 seconds. If the concentration of
your sample is very low, you should use a 1:1 v/v matrix/analyte mixture.
4) Transfer 1  of the analyte/matrix solution on the sample plate and allow it to
completely dry. If you are going to use a 1:1 v/v matrix/analyte solution, you may spot
them on the plate separately. Place 1  of the analyte solution on the plate,
immediately followed by 1  of the matrix solution.* Load the sample plate.
*Placing the matrix on the plate first, will cause it to crystallize before you add the
analyte. This will prevent a homogeneous matrix/analyte crystallization.
Glycoproteins
Glycoproteins also work well with CHCA and SA, depending on the mass. Slightly
higher concentrations are recommended. SA works well with the dried-droplet method.
For smaller glycoproteins, < 5000 DA, CHCA (and DHB) usually work well. In
addition to the dried-droplet method, the sandwich method, and the fast-evaporation
method have also been recommended. Unfortunately, experiments have shown that
there is no particular method that works well for all glycoproteins/glycopeptides and
each sample has to be treated individually. THAP also works well for glycopeptides,
especially acidic glycopeptides. HABA has also been suggested to work as well as SA
(or even better than SA) for larger glycoproteins. The mixture of the additive 5methoxysalycilic acid (2-hydroxy-5-methoxybenzoic acid) with DHB (called super
DHB) has also proven to be a good matrix for glycoproteins as well as for
carbohydrates.
Hydrophobic Proteins
Since detergents and organic solvents are needed for the extraction of hydrophobic
proteins (membrane proteins), the solubility of these compounds is different from other
classes of proteins. However, once solubilization of matrix and protein has been
achieved, the normal sample preparation techniques works well for hydrophobic
proteins. CHCA, SA and DHB work well for hydrophobic peptides/proteins. They can
be dissolved in organic solvents such as ACN in various ratios such as 1:2 v/v with
0.1%TFA. Also, if your analyte is dissolved in chloroform, you can dissolve your
matrix in a 2:1 v/v chloroform/methanol solution. HABA dissolved in ACN/0.1%
TFA(2:1 v/v) has also been used in the analysis of hydrophobic compounds. The
addition of formic acid to the solvent used for the preparation of the matrix (70%
aqueous solution) has also been suggested. In this case, your analyte is dissolved in a
solution of formic acid/hexafluoro-2-propanol (10 mM). It is important to note that the
formylation of peptides has been reported in the presence of formic acid and
therefore, %0.1TFA is used more often than formic acid.
Sample Preparation for Oligonucleotides
If your sample is a complete unknown (you have no idea what the mass is) you may
want to start with HPA. Usually all oligonucleotides give a MALDI signal with HPA.
Then you can optimize the conditions depending on the peaks you observe in the
spectrum. The general rule for this class of samples is based on their mass. For
compounds less than ~3500 Da in mass, THAP works best and for masses higher than
that, HPA is the matrix of choice. Some groups still use DHB for smaller
oligonucleotides, but the most commonly used matrix for this class of compound is
THAP. You will find the protocols for sample preparation for oligonucleotides below
but for a detailed graphical presentation of the sample preparation procedure read:
"MALDI in the Lab":
1) Adjust the concentration of your sample. The optimum concentration for
oligonucleotides is between 50-100 pmole/ L (this will give a final concentration of 510 pmole/L after mixing with the matrix). If the concentration is unknown make a
series of dilution in deionized H2O. Tip: Do not use HPLC grade water for
oligonucleotides as it can contain different concentrations of salts.
2) Prepare a 0.025 M solution of diammonium citrate* in 1:1 v/v ACN/H2O and
dissolve THAP or HPA in this solution until you arrive to a saturated solution of matrix
in the diammonium citrate solution. Vortex the mixture for 60 seconds and then
centrifuge it for 20 seconds. Use only the supernatant solution. This is to prevent any
inhomogeniety in the crystallization, since any particulate in the solution can act as a
nucleation site and cause inhomogeniety in the crystallization.
3) Transfer 9 L of the matrix solution to a 0.5 ml eppendorf tube. Add 1 L of the
analyte and either mix with the tip of your pipette tip or vortex gently for 5 seconds.
Keep in mind that for samples with concentrations below 50 pmole/L, you should use
less matrix in the mixture to keep the final analyte concentration in the appropriate
range (5-10 pmole/L). A 1:1 v/v analyte/matrix ratio is recommended for low
concentration samples.
4) Transfer 1L of the analyte/matrix solution on the sample plate and allow it to
completely dry. If you will be using a 1:1 v/v matrix/analyte solution, you may either
use the mixture or place them on the plate separately. In this case, transfer 1 L of the
analyte directly on the sample plate followed by 1 L of the matrix.** Allow the sample
to dry. Load the sample plate
*In the preparation of oligonucleotides it is common to add an ammonium salt to the
matrix solution. The exchange of NH4+ with the cations such as Na+ and K+ greatly
enhances the resolution by ridding the sample from salt adducts. NH4+ is released
during the desorption process in the form of ammonia and does not appear in the
spectrum.
**Placing the matrix on the plate first, will cause it to crystallize before you add the
analyte. This will prevent a homogeneous matrix/analyte crystallization.
Sample Preparation for Synthetic Polymers
Since DHB works well for a large number of polar and non-polar samples, it is a good
matrix to start with for polymers. You can then optimize the conditions depending on
the peaks you observe in the spectrum. DIT and IAA are other matrices that are
frequently used for the analysis of polymers. DIT works well with aromatic polymers
and also for non-polar polymers. IAA has been known to work best for non-polar
polymers. The general rule for this class of compounds is to first figure out the solvent
in which the analyte dissolves in. Since one of the criteria of a good matrix is dissolving
in a solvent that is miscible with the solvent of the sample, this will give you some idea
of where to start. Below are some general rules that will guide you through making the
right choice (for a detailed graphical presentation of the sample procedure read:
"MALDI in the Lab").
1) Adjust the concentration of your sample. The optimum concentration for polymers is
~10-3 M. Note that this is significantly higher than peptides/proteins and
oligonucleotides. After mixing with the matrix, this will give you a final concentration
of ~10-4M. If the concentration is unknown make a series of dilution in the solvent of
choice.
2) Prepare a saturated solution of the matrix in an appropriate solvent. This should be a
solvent that is miscible with the solvent of the analyte. Vortex the mixture for 60
seconds and then centrifuge it for 20 seconds. Use only the supernatant solution. This is
to prevent any inhomogeniety in the crystallization, since any particulate in the solution
can act as a nucleation site and cause inhomogeniety in the crystallization.
3) Transfer 9 L of the matrix solution to a 0.5 ml eppendorf tube. Add 1 L of the
analyte and either mix them with the tip of your pipette tip or vortex for 5 seconds.
Keep in mind that if your sample is very dilute (conc. < 10-4 M), you should mix your
polymer with a smaller volume of the matrix. A 1:1 v/v is a good ratio to start with.
4) Transfer 1L of the analyte/matrix solution on the sample plate and allow it to
completely dry. If the solvent you have used has a low surface tension, the sample will
spread over the plate. If you are spotting more than one sample on the plate, leave one
spot between the samples to avoid spill overs from one well to another. Load the sample
plate.
Sample Preparation for Organic Molecules
The analysis of organic molecules depends in part to the solvent in which the analyte is
soluble in. For very small molecules, the mass of the analyte also plays a role in
choosing the matrix. If you have a molecule with a mass of 379 Da, you can not use
CHCA as the matrix since a strong peak from the dimer of CHCA appears at this mass.
DHB usually works well for organic molecules. Being soluble in a number of solvents,
such as H2O, THF, methanol, acetone, and ACN makes it a good candidate for many
organic molecules. DIT has been known to work well with samples such as fullerenes
and porphyrins. DIT dissolves well in acetone, toluene, CH2Cl2, and other non-polar
solvents. It can be used in cases that the solvent of the analyte is not miscible with that
of DHB. The sample preparation protocol most commonly used for organic molecules
follows (for a detailed graphical presentation of the sample procedure read: "MALDI in
the Lab").
1) Adjust the concentration of your sample. The optimum concentration for small
organic molecules varies greatly depending on the sample. A good concentration to start
with is ~ 200 pmole/L (this will give a final concentration of 20 pmole/L after
mixing with the matrix). If the mass of the analyte is smaller than 500 Da, you may
need to increase the concentration. This is due to the fact that strong matrix peaks are
observed below 500 Da.
2) Prepare a saturated solution of DHB in a solvent that is miscible with that of the
analyte. Vortex the mixture for 60 second and then centrifuge it for 20 seconds. Use
only the supernatant solution. This is to prevent any inhomogeneity in the crystallization,
since any particulate in the solution can act as a nucleation site and cause
inhomogeneity in the crystallization.
3) Transfer 9 L of the matrix to a 0.5 ml eppendorf tube. Add 1 L of the analyte and
either mix with the tip of your pipette tip or vortex for 5 seconds. If the concentration of
your sample is unknown, you can start with a 1:1 v/v analyte/matrix solution.
4) Transfer 1L of the analyte/matrix solution on the sample plate and allow it to
completely dry. Load the sample plate.
Sample Preparation for Carbohydrates
The most popular matrix used today for carbohydrates is DHB. The [M+Na]+ is the
major ion observed in the MALDI spectra of carbohydrates which is accompanied with
the [M+K]+ ion. The mixture of DHB with additives can improve the resolution and
sensitivity. Among these additives 5-methoxysalycilic acid (2-hydroxy-5methoxybenzoicacid) has given the best results. This mixture known as "Super DHB"
also works well for glycoproteins. CHCA works well for smaller carbohydrates (< 2000
Da). THAP is a neutral matrix and if you are analyzing an acidic carbohydrate, it is
better to use THAP. For a graphical presentation of the sample preparation procedure
read "MALDI in the Lab".
1) Adjust the concentration of your sample. The optimum concentration for
carbohydrates is between 100-150 pmole/L (this will give a final concentration of 1015 pmole/L after mixing with the matrix). If the concentration is unknown make a
series of dilutions in H2O.
2) Prepare a saturated solution of DHB in H2O. Vortex the mixture for 60 seconds and
then centrifuge it for 20 seconds. Use only the supernatant solution. This is to prevent
any inhomogeniety in the crystallization, since any particulate in the solution can act as
a nucleation site and cause inhomogeniety in the crystallization. DHB has less solubility
in H2O than in some organic solvents such as methanol or ACN. You can add a small
amount of ACN to the solution to form a mixture (1:2 v/v ACN/H2O works well).
CHCA can be dissolved in a 1:1 v/v ACN/0.1% TFA. THAP works well with 1:1 v/v
ACN/H2O.
3) Transfer 9 L of the matrix to a 0.5 ml eppendorf tube. Add 1 L of the analyte and
either mix with the tip of your pipette tip or vortex for 5 seconds. Keep in mind that if
your analyte has a lower concentration than mentioned above (<100-150 pmole/L),
you should mix your analyte with a smaller amount of matrix solution. If you have a
very dilute solution you may want to add only 1 L of matrix to your analyte.
4) Transfer 1 L of the analyte/matrix solution on the sample plate and allow it to
completely dry. Load the sample plate.