Online Inquiry

Protocols for Identification of Proteins by MALDI-TOF MS

1. Sample Preparation for Peptides/Proteins

1) Adjust the concentration of the sample. The optimal concentration of peptides and proteins is 5-50 pmol/μL, with a final concentration of 0.5-5 pmol/μL after mixing with the matrix.

2) If the concentration is unknown, make a series of dilutions in 0.1%TFA to produce a series of sample spots on the MALDI plate.

3) If sample is very dilute, concentrate by Speed-Vac or pipet-tip chromatography.

4) Prepare a saturated solution of the matrix in a 1:1 v/v ACN/0.1%TFA solution. Add the aqueous TFA solution, then the ACN. Vortex the mixture for 60 s, then centrifuge for 20 s. Use only the supernatant, since any particulates in the solution can act as nucleation sites and cause inhomogeneity in the crystals.

5) Spot the matrix and the sample on the plate separately by placing 0.5 μL of the analyte solution on the plate, immediately followed by 0.5 μL of the matrix solution. Do not touch the surface of the plate with the pipet tip, otherwise uneven crystallization will occur.

6) Allow to air-dry. The mixture forms a co-crystalline sample/matrix complex.

7) Alternatively, mix the sample in solution (1–10 pmol/μL) with an excess of the matrix (in a ratio between 1000:1 and 10,000:1) in a 0.5-mL Eppendorf tube. Mix with the pipet tip or vortex for 5 s. Transfer 1 μL of this solution to the sample plate and allow to dry completely.

8) Place the target plate containing all the samples in the instrument and evacuate. Pulses of laser light are applied, which causes rapid excitation and vaporization of the crystalline matrix and subsequent ejection into a plume of matrix and analyte ions, which are analyzed by their time of flight.

9) With the aid of the camera that is used to follow the exact position of the laser flash, move the laser beam around the MALDI plate to find so-called sweet spots, where the composition of co-crystallized matrix and sample is optimal, in order to obtain good sensitivity.

10) Once a good quality signal has been obtained by optimizing the parameters, a few hundred (typically) pulses of laser light are used to accumulate sufficient signals to generate a good mass/charge (m/z) spectrum. The laser can be tracked cross the circular spot automatically by a program (a macro) during this time to cover the complete area of the spot, in order to ensure that the sample/matrix sweet spots are targeted. The tracking can be lateral across and down the spot or in a spiral, for example.

11) Calibrate the mass in the spectrum externally or internally using appropriate molecular weight standards analyzed under similar conditions.

2 On-Plate Washing to Remove Buffer and Salts

If there are known (or suspected) high levels of contaminant that may cause problems with the analysis, the dried crystalline sample on the target plate may be washed with a few μL of water or 0.1% aqueous TFA, provided the matrix is not highly water soluble. This simple washing step frequently results in a dramatic improvement in the spectral data. If the sample is sufficiently concentrated, it can simply be diluted to minimize interference from contaminating substances.

1) Dry sample and matrix.
2) Deposit 1–2 μL of cold high-purity water containing 0.1% TFA on the sample spot.
3) Leave on for 5–10 s, then remove.
4) Repeat the wash step as necessary.
5) To reduce or remove detergent contamination, use a similar wash step(s) with 5% aqueous propan-2-ol.
6) To reduce contamination from crude cell extract, use 100% propan-2-ol.


  1. Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
* For Research Use Only. Not for use in diagnostic procedures.
Our customer service representatives are available 24 hours a day, 7 days a week. Inquiry

Online Inquiry

Please submit a detailed description of your project. We will provide you with a customized project plan to meet your research requests. You can also send emails directly to for inquiries.

* Email
* Service & Products of Interest
Services Required and Project Description
* Verification Code
Verification Code