This section includes some advices/ recommendations in order to make our service as effective as possible:
> Contaminations
> Reagents compatible with MALDI-TOF mass spectrometry
> Source of sample
> Amount of sample
> Shipment of sample
Contaminations
In order to avoid contaminations, the cleaning of the used material is fundamental along sample preparation. Use material rinsed with Milli-Q water (not autoclaved). It is highly recommended the use of gloves during all the procedure to minimize the risk of contamination with other proteins such as keratines. These keratines are present in the hair, the skin and in certain types of clothes, making difficult the identification of the protein of interest
Compatible reagents with MALDI-TOF Mass Spectrometry
Use reagents of HPLC-grade and Milli-Q water.
* Reagents that do not interfere with mass spectrometry:
Trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, b-mercaptoethanol, DTT, volatile organic solvents, sodium hydroxide.
* Reagents that do not interfere with mass spectrometry when their concentration is lesser than 50 mM:
HEPES, MOPS, Tris, sodium hydroxide, octyl- glucoside acid.
Urea and guanidine do not interfere up to 2M.
* Avoid the following compounds:
glycerol, sodium azide, DMSO, SDS, phosphates, NaCl.
Source of sample
The sample can be a protein isolated from a SDS-PAGE gel (a monodimensional separation provides a band) or from a 2D gel (a bidimensional separation provides a spot). The gel staining can be performed with Coomassie Blue dye or with a silver solution compatible with mass spectrometry (the fixing to the gel is reversible -see protocols).
Amount of sample
The minimum peptide or protein concentration needed for mass spectrometry analysis is in the range 5 fmol/ µl - 1 pmol/µl. (1 pmol/µl = 10 -6 M).
This amount is similar to detected protein concentration by Coomassie blue staining and equivalent to the most part of the proteins detected by silver staining
Shipment of sample
If the sample comes from a polyacrilamide gel, cut the protein spots (bidimensional gel) avoiding unnecessary amounts of the gel and/or protein mixtures. It can be carried out with the plastic tip of a pipette previously cut (approximately 1.5 mm 2 ). The bands of a monodimensional gel can be cut with a clean scalpel.
It is necessary to send an image of the gel (in a file or a hardcopy).
Introduce the piece of the gel in an eppendorf tube and add Milli-Q water
