MALDI Sample Preparation: the Ultra Thin Layer Method


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This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

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Fenyo, D., Wang, Q., DeGrasse, J. A., Padovan, J. C., Cadene, M., Chait, B. T. MALDI Sample Preparation: the Ultra Thin Layer Method. J. Vis. Exp. (3), e192, doi:10.3791/192 (2007).


This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.


It is very important to wear powder-free gloves during the preparation of the thin layer.

Cleaning of the sample plate

  1. Use a stainless steel or gold MALDI sample plate.
  2. Wash with MeOH and wipe gently with a Kimwipe. Do not rub or scrub the surface with the Kimwipe, to prevent scratching the surface.
  3. Wash with H2O and wipe gently with a Kimwipe.
  4. Wash with MeOH and wipe gently with a Kimwipe.
  5. If needed, repeat MeOH/H2O/MeOH cleaning cycle, always ending with MeOH.
  6. Make sure that there are no ghost spots or residue remaining on the plate. If there are ghost spots, rinse the plate with deionized water while rubbing the surface with your gloved hand (Do not rub with Kimwipes). Repeat the MeOH/H2O/MeOH cleaning cycle.
  7. Use the plate immediately after washing.

Preparation of the thin layer substrate solution

  1. Mix 1 part of saturated 4-HCCA (2 parts ACN, 1 part water, and 0.1% final TFA) and 3 parts isopropanol.

    Note: The thin layer substrate solution is very stable, and can be kept at room temperature in the dark for a year. It may also be stored at 4 °C.

Making the thin layer substrate

  1. Apply 20-50 µL of the thin layer substrate solution on the left-center of the plate, and spread with the side of a pipette tip. Sweep in one direction.
  2. As the solution dries, the organic solvent evaporates very quickly, leaving behind minute droplets of water on the surface of the plate. At this point, wrap your index finger with a ply of Kimwipe and initially gently blot the surface of the plate with it.
  3. When the surface is freed from water droplets, wipe the entire plate with uni-directional sweeping motions using a Kimwipe.

    Note: If the solution does not spread enough throughout the plate surface, increase the ACN composition and recreate the thin layer. Some parameters that may affect the spreading of the solution include ambient humidity and temperature.

    Note: With gold plates, the layer usually appears as a yellowish reflection, depending on viewing and light angles. With steel plates, only the edge of the layer is normally visible.
  4. Clean the edges of the plate with a fresh Kimwipe before placing it in the plate holder to prevent matrix from contaminating the instrument.

Preparation of the matrix solution

  1. Saturated 4-HCCA in FWI (3 parts formic acid, 1 part water, 2 parts isopropanol) is recommended for analysis of both membrane and soluble proteins. It might also be used in special cases for the analysis of very hydrophobic large peptides (M >4,000 u). Make sure that the matrix solution used for spotting the proteins does not exceed ~50% organic content, as it will completely dissolve the thin layer substrate, defeating the advantages of the method.

Test of the thin layer substrate

  1. Spot 0.5 µL of the matrix solution on the plate. A whitish precipitate will appear at the interface between the thin layer and the droplet. When this layer covers the bottom of the droplet entirely, usually within 10-15 sec, aspirate the excess liquid with a vacuum line. If it takes longer than 30 sec for the precipitate to form, it is an indication that there might be a problem with the thin layer substrate and/or the matrix solution.

Sample application

  1. The analyte concentration in the starting solutions should be within 10 and 1000 µM.
  2. Dilute the analyte solution in matrix solution to a final analyte concentration between 0.2–2 µM. For example, start with 1 µL of sample and 9 µL of matrix solution.

    Note: Make sure that the matrix is close to being saturated in the final solution, otherwise you will redissolve the thin layer substrate.

    Note: Analyte concentrations required to obtain decent signals are highly dependant on solution complexity and composition (e.g., contaminants) and analyte ionizability.

    Note: If a single-step dilution does not yield good results, you should try another dilution step. Although counter-intuitive, highly concentrated analyte solutions rarely produce good spectra.

    Note: Membrane proteins usually require larger dilutions.
  3. Spot 0.5 µL of sample/matrix mixture on the plate. A whitish precipitate will form at the interface between the thin layer and the droplet. This precipitate is the cocrystallization of matrix and analyte. When this layer covers the bottom of the droplet entirely, usually within 10-15 sec, aspirate the excess liquid with a vacuum line. If it takes longer than 30 sec for the precipitate to form, it may be an indication of a contaminant present, which prevents crystallization, or your analyte is too concentrated.

Calibrant application

  1. Prepare the calibrant/matrix mixture in a similar manner with a final calibrant concentration between 0.1–0.5 µM.
  2. Spot 0.5 µL calibrant/matrix mixture on the plate in close proximity to the sample spots. This physical proximity is used in a pseudo-internal calibration procedure in which the plate is moved from the sample spot to the calibrant spot during sample acquisition, to add a few laser shots of the calibrants to the spectrum. This approach guarantees a higher mass accuracy calibration than external calibration only.

Washing step

  1. Wash each spot with approximately 2 µL of a ice cold 0.1% aqueous TFA solution. Aspirate excess liquid with a vacuum line.

Acquisition of mass spectra

  1. You are now ready to acquire mass spectra. If you use the FWI matrix solution, you must acquire your spectra as soon as possible to prevent irreversible modification to the analyte such as formylation.

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  1. Xiang, F., Beavis, R. C. A Method to Increase Contaminant Tolerance in Protein Matrix-Assisted Laser Desorption Ionization by the Fabrication of Thin Protein-Doped Polycrystalline Films. Rapid Communications in Mass Spectrometry. 8, 199-204 (1994).
  2. Cadene, M., Chait, B. T. A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins. Analytical Chemistry. 72, 5655-5658 (2000).
  3. Hook, P., Mikami, A., Shafer, B., Chait, B. T., Rosenfeld, S. S., Vallee, R. B. Long-range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains. J Biol Chem. 280, 33045-33054 (2005).




    Posted by: Anonymous
    December 15, 2008 - 1:34 AM
  2. MALDI, especially with the ultra thin layer method described here, can be use to reliably measure the molecular masses of small quantities of proteins (or protein fragments), even if they are relatively insoluble. For example, the described method allows for the mass spectrometric analysis of intact membrane proteins that are only soluble in the presence of detergents and/or membrane lipids. We have found the procedure to be highly robust for proteins ranging in molecular mass up to as high as 380 kDa. When using electrospray ionization, such measurements can often be quite challenging, especially when only small amounts of protein (sub-picomole) are available and extensive optimization of conditions is not feasible. Mass accuracy in the 100 ppm range is possible in cases where the protein of interest is highly homogenous (see reference ² above).

    Posted by: Anonymous
    June 23, 2009 - 5:38 PM
  3. Congratulations for this excellent method. I have a doubt about the formic acid concentration. Reference ² describ formic acid reagent at 88% and I think that it was dilute to 1%. Am I right?

    Posted by: Anonymous
    January 12, 2010 - 3:38 PM
  4. The reagent is indeed Formic Acid, 88% (Certified ACS), Fisher Chemical, as stated in reference ². I think, strictly speaking, the specification that Fisher gives is >=88%.

    Posted by: Anonymous
    January 12, 2010 - 3:51 PM

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