Users are strongly encouraged to consult with MS facility personnel before submitting a sample for analysis.
Samples in solution must be clearly labeled and should be submitted in 0. 5 ml or smaller Eppendorf-type tubes. Although purity is not a requirement for LC/MS and ESI, samples should be solvent-free and if possible in water. Contaminants present in the sample could supress the signal of the protein of interest or they can make the spectra difiicult to interpret.
All samples must be accompanied by a sample submission form with detailed information of the sample submitted. When the samples are from SDS-PAGE, include a picture of the Coomasie stained gel.
Protein Solutions for Gel-Free Analysis
Protein solutions should be frozen at -80°C as soon as possible and shipped to us on dry ice.
Note about proteins and peptides: Proteins and peptides in solution should not be stored in glass vials. It is our experience that proteins will adsorb irreversibly onto the glass. Samples in solution should be stored in 0.25 or 0.5 ml Eppendorf-type centrifuge tubes.
In-Gel digests and Protein ID by MS. The facility offers automatic sample processing of 1-D and 2-D gels, in preparation for analysis by MS.
Gel bands from 1D gel electrophoresis or gel spots obtained from 2D gel approaches should be cut with the following considerations in mind:
The excised gel material should be placed in individual 0.25 or 0.5 ml microcentrifuge tubes or a 96 well plate. We could provide you with 96 well plates that are custom built for our digestion robot, which will reduce sample handling and reduce potential sources of contamination.
Detergents and Mass Spectrometry1
Please talk to us about any detergents that are used in your sample preparation procedure, even if they are far upstream in your sample handling protocol.
Unfortunately, most detergents are not compatible with downstream mass spectrometry analysis on the LTQ FT. Dilution, washing, and detergent removal columns cannot remove enough detergent for successful analysis of your sample or to prevent significant contamination of the mass spectrometer. Detergent contamination of a mass spectrometer is very costly (ruined columns/tubing) and very time consuming to the laboratory. If your samples are very complex, it might be possible to purify them by running them through an SDS-PAGE gel.
Some detergents can be separated from the sample by standard SDS-PAGE or TCA - cold acetone precipitation. However, Triton-X and Tween-20 cannot be used under any circumstances. These cannot be removed from your sample using dilution, washing, detergent spin columns, or SDS-PAGE.
Other Mass Spec Friendly Detergents
MALDI-MS and MALDI-MS/MS For successful evaluation and acquisition of the MALDI-MS and MALDI-MS/MS spectra the following criteria for the peaks of interest need to be fulfilled (Trypsin autoproteolytic peptides are not evaluated.):
The internal calibration is performed using porcine trypsin (Promega) auto-proteolytic peptides at m/z 842.5099; m/z 1045.5642, and m/z 2211.1046, or in the event that these peptides are not detectable, using the external calibrant spotted in close proximity to the sample spot. The resulting mass accuracy by external or internal calibration is better than 50 or 20 ppm, respectively for the MS, and within 200 ppm for the MS/MS mode.
ESI-MS and ESI-MS/MS. ESI-MS analysis will be performed on a LTQ-FT mass spectrometer. MS/MS will be done automatically under software control. In general, duplicate MS/MS will be acquired on the three to six most intense ions detected on the previous full-MS scan; ion exclusion will be turned on for 90 seconds. Background ions (i.e. calibration standards, tryptic peptides...) will be excluded. Mass accuracy can be as good as 2 ppm, depending on detector settings.