Abbreviations: alphaCHCA= alpha cyanohydroxycinnamic acid, SA= sinapinic acid, DHB= dihydroxybenzoïc acide, TFA= trifluoroacétique acid, aq.=aqueous.
Products References: alphaCHCA, SA, DHB, nitrocellulose
 HPLC ou sequencing grade solvents
General Remarque : Solutions must be prepared extemporaneously. Samples can be dried on the MALDI target under slight vaccum 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 (uncolored, 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 (uncolored, clear): nitrocellulose 40mg.ml^-1 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 solvants, 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 labeled 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.

References:
    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 charaterization 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.
 
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Material: Gel loader tips 1-10 µL Eppendorf, Poros R2 50 phase (PerSeptive Biosystems), microtubes 1.5mL Treff

Prepare a support with two holes in the cover of a 1.5mL microtube. The gel loader tip should be hold in it with the end of the tip in contact with the bottom of the microtube.
Pinch the narrower part of the tip to reduce the internal diameter. Add 30µL of methanol in the tip, then 2.5µL of Poros phase in suspension in methanol, and finally 30µL methanol more. Avoid bubbles. Let the phase precipate in the tip for 30s-1mn. Centrifuge for a few seconds. If necessary cut the final part of the tip.
Add 30µL of aq. formic acid 5%, centrifuge for a few seconds to rince the phase without drying it.
Add 30µL of aq. formic acid in the dried sample, vortex well, centrifuge. Add this solution in the tip, centrifuge for a few seconds.
Rince the phase with 20µL of aq. formic acid 5% three times.
Change the 1.5mL microtube, and eluate the compounds by adding twice 4µL methanol.
The sample in methanol can be directly analysed or reconcentrated under vacuum, if it is too diluted.

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