Sample preparation

Sample preparation for mass spectrometry

Samples can be prepared as either 1D bands or 2D gel spots. Depending on the amount of material they can be stained with standard Coomassie Blue. Alternatively samples with much lower abundance can be stained with Sypro Ruby (a fluorescent stain from Molecular Probes) or Silver. Silver staining must omit the use of Glutaraldehyde in the fixing process. Most modern kits from BioRad, and Pierce are compatible with mass spectrometry. It is extremely important to remember that when preparing low abundance proteins precautions must be taken to prevent keratin contamination. This means wearing gloves during the entire preparation process and using a clean apparatus for running gels etc. When excising bands of interest cut as closely to the staining boundary as possible because the resolution of your gel is only as good as how you cut.

SAMPLE PREPARATION FOR MALDI

Tip and tricks for MALDI sample preparation

!!!!Avoid non volatile solvents!!!!! Several involatile solvents are common in protein chemistry: glycerol, polyethylene glycol, b-mercaptoethanol, Triton-X100, DMSO, DMF, etc. These solvents interfere with matrix crystallization and coat the crystals that are formed. Try the crushed-crystal method if signal suppression due to involatile solvents is suspected.

• Dried MALDI samples are quite stable, they can be kept in a dark drawer, in the refrigerator/freezer, or in a vacuum for days. It is common practice in many analytical labs to freeze samples indefinitely in sealed containers filled with inert gas.


• The analyte must be soluble in the final analyte/matrix mixture solvent. The matrix and the analyte are often dissolved in different solvents and they must all remain in solution after they are mixed together. Centrifuge if necessary, and use the supernatant.


• Beware of changes in solvent composition during sample preparation as caused by selective evaporation of volatile organic solvents from aqueous solutions. This is facilitated by the small sample volumes used.


• Do not heat analyte/matrix droplets to speed drying. Changing the solution's temperature alters matrix crystal formation and protein incorporation. It usually makes things worse.


• Use fresh matrix solutions. Mix up small volumes of solution as needed, and date them. Do not use the same solutions for more than two or three days. To be safe, prepare new solutions every day!


• If you suspect that contaminants are suppressing your signal, try to purify the sample prior to MALDI analysis. There are many convenient sample purification procedures perfectly fit for MALDI sample purification. If the MALDI deposit is already made, wash the crystals with cold water and retry the MALDI measurements before giving up.


• It is good practice to wash metal sample substrates between runs. When washing your plates, first rinse your plate with the same solvent that your last sample was dissolved in. For example, if you were analyzing a polymer soluble in methanol with DHB as the matrix, first wash your plate with methanol to make sure you have rinsed off your sample and no contaminants will be carried over to the next set of samples you will be analyzing. Then, wash the target with a very dilute detergent solution and a very soft toothbrush, rinse thoroughly with DI water and let it completely dry before using it again. For this reason, it is always good to have spare sample plates.


• Do not leave undissolved matrix crystals in the final analyte/matrix solution. Intact crystals can act as nucleation sites, leading to very inhomogeneous crystals. If necessary centrifuge the final solution and use only the supernatant.


• The crystal growing solution's pH must be less than 4. Most organic acids used as matrices dissociate at pH>4, and that seriously affects the outcome of crystallization. Using aqueous 0.1%TFA (v/v in H₂O) rather than straight water usually eliminates any pH concerns.


• Remember that contrary to what you would expect : reducing the analyte concentration is usually what makes the MALDI signal go up! Do dilution series.


• Sample preparation procedures that premix the sample with the matrix usually yield more reproducible results for mixtures.


• The acetone redeposition technique is a trick often used by MALDI practitioners to improve sample homogeneity. In this technique, the dried MALDI sample prepared by any of the procedures discussed in the crystallization methods, is redissolved in a single drop of acetone. As the acetone evaporates, the sample recrystallizes to form a more homogeneous film, that leads to increased spot-to-spot reproducibility.

Deposition methods

Abbreviations: alphaCHCA= alpha cyanohydroxycinnamic acid, SA= sinapinic acid, DHB= dihydroxybenzoic acid, TFA= trifluoroacetic acid, aq.=aqueous. Products References: alphaCHCA, SA, DHB, nitrocellulose HPLC or sequencing grade solvents.

General Remarque: Solutions must be prepared extemporaneously. Samples can be dried on the MALDI target under slight vacuum in a dessicator for example.

a. "Dried Droplet" preparation alphaCHCA
Prepare a solution of saturated alphaCHCA in (aq. TFA 0.1%)/acetonitrile 1:1 (v/v). (yellow) Spot on the target the same volume of the matrix solution and of the sample solution (about 1µL), mix well and let it dry.

b. "Dried Droplet" preparation SA
Prepare a solution of saturated SA in (aq. TFA 0.1%)/acetonitrile 1:1 (v/v). (white). Spot on the target the same volume of the matrix solution and of the sample solution (about 1µL), mix well and let it dry.

c. "Dried Droplet" preparation DHB
Prepare a solution of saturated DHB in (aq. TFA 0.1%)/ethanol 9:1 (v/v)(white). Spot on the target the same volume of the matrix solution and of the sample solution (about 1µL), mix well and let it dry.

d. Thin Layer preparation alphaCHCA/nitrocellulose
Prepare a solution A (uncoloured, clear): nitrocellulose 10mg.ml-1 in acetone/isopropanol 1:1 (v/v).
Prepare a solution B (yellow): alphaCHCA saturated in acetone.
Prepare a solution C: mix A/B 1:4 (v/v).
Spot 1µL of C, let it dry (almost instantaneous).
Spot 0,5 to 1µL of the acidic solution of peptide (TFA 0.1%).
Let it dry.
(Wash once or twice with 5µL of aq. TFA 0,1%, let it dry between each washing step).

e. Sandwich preparation SA/alphaCHCA
Prepare a solution A (uncoloured, clear): nitrocellulose 40mg/ml in acetone.
Prepare a solution B (yellow): alphaCHCA saturated in acetone.
Prepare a solution C: mix A/B/isopropanol 1:1:2 (v/v).
Prepare a solution D (white): SA in (aq. TFA 0.1% )/acetonitrile 1:1 (v/v).

Spot 1 µL of C, let it dry.
Spot 1 µL of the acidic solution of peptide (TFA 0.1%), let it dry.
Spot 1 µL of D, let it dry.
(Wash once if necessary just before the total evaporation of solvents, after the beginning of crystallisation)

f. Single Cell Analysis
Sample preparation for MALDI after fixation and immunodetection. Cells previously characterized by immunohistochemistry were dissociated by adding trypsin to the PBS buffer for 30 minutes at room temperature. After manual dissociation, the labelled cells were aspired into a thin glass capillary and deposited on the stainless steel target. The excess of PBS was removed with the same capillary. After a quick wash with 1 µL of a saturated solution of DHB in 0.1% TFA, a 0.5 µL drop of the same matrix solution was added and dried at room temperature. A second 0.5 µL drop of matrix was then added and dried in the same way. The target was then immediately inserted in the mass spectrometer.

• Kussman, M., E. Nordhoff, et al. (1997). Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Sample Preparation techniques Designed for Various Peptide and Protein Analytes. Journal of Mass Spectrometry 32: 593-601.


• Cohen, S. L. and B. T. Chait (1997). Mass Spectrometry of Whole Proteins Eluted from Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Gels. Analytical Biochemistry 247(2): 257-267.


• Mann, M. and G. Talbo (1996). Developments in matrix-assisted laser desorption/ionization Peptide Mass Spectrometry. Current Opin. Biotechnol 7: 11-19.


• Vinh, J., D. Loyaux, et al. (1997). Sequencing branched peptides with CID/PSD MALDI-TOF in the low-picomole range: application to the structural study of the posttranslational polyglycylation of tubulin. Analytical Chemistry 69(19): 3979-85.


• Gevaert, K., H. Demos, et al. (1998). A peptide concentration and purification method for protein characterization in the sub-picomole range using matrix assisted laser desorption/ionization - post source decay (MALDI-PSD) sequencing. Electrophoresis 19.


• van Veelen, P. A., C. R. Jimenez, et al. (1993). Direct Peptide profiling of Single Neurons by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. Organic Mass Spectrometry 28: 1542-1546.


• Garden, R. W., L. L. Moroz, et al. (1996). Excess Salt Removal with Matrix Rinsing: Direst Peptide profiling of Neuron from Marine Invertebrates Using Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry. Journal of Mass Spectrometry 31: 1126-1130.


• Redeker, V., J. Y. Toullec, et al. (1998). Combination of peptide profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunodetection on single glands or cells. Anal Chem 70(9): 1805-11.