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/mL (this will give a final concentration of 0.5-5 pmole/ml 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 ml of the matrix to a 0.5 ml eppendorf tube. Add 1 ml 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 ml 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 ml of the analyte solution on the plate, immediately followed by 1 ml 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 5-methoxysalycilic 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/m L (this will give a final concentration of 5-10 pmole/ mL 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 mL of the matrix solution to a 0.5 ml eppendorf tube. Add 1 mL 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/mL, you should use less matrix in the mixture to keep the final analyte concentration in the appropriate range (5-10 pmole/mL). A 1:1 v/v analyte/matrix ratio is recommended for low concentration samples.

4) Transfer 1mL 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 mL of the analyte directly on the sample plate followed by 1 mL 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 mL of the matrix solution to a 0.5 ml eppendorf tube. Add 1 mL 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 1mL 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/mL (this will give a final concentration of 20 pmole/mL 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 mL of the matrix to a 0.5 ml eppendorf tube. Add 1 mL 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 1mL 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-5-methoxybenzoicacid) 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/mL (this will give a final concentration of 10-15 pmole/mL 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 mL of the matrix to a 0.5 ml eppendorf tube. Add 1 mL 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/mL), 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 mL of matrix to your analyte.

4) Transfer 1 mL of the analyte/matrix solution on the sample plate and allow it to completely dry. Load the sample plate.