Sample Preparation for Submission

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:

  1. Beware of contaminating proteins in all phases of sample handling. Dust particles contain high levels of proteinaceous material, especially skin and hair proteins of which the members of the keratin class are the most abundant. As early as possible, in the sample preparation, step "anti-dust" measures should be introduced. These measures include thoroughly washing all glassware and plastic ware, having dedicated "mass spectrometry" solutions that have been filtered, always wearing gloves, lab coats, and covering the gel through all phases of staining and destaining. When we engage in manual gel cutting, we perform this step in a laminar flow hood to reduce the possibility of dust contamination.
  2. Try to keep the volume of gel matrix material to a minimum. Excess amounts of polyacrylamide will reduce the yield of identifiable peptides from a band or spot. Try to cut away as much unstained gel material as possible.
  3. Keeping the protein contained within the gel slice/band slightly acidified reduces the activity of the alkaline proteases. We typically store our gel bands/spots at 4°C in 1% acetic acid.
  4. Do not freeze the gel bands/spots since thawing gel bands that have previously been frozen results in leakage of intact proteins from the gel matrix.

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 by ESI mass spectrometry. 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.

In-solution Digestion

  • Compatible Detergents
    • 0.05%-1% SDS
    • 0.05%-0.5% CHAPS (although not recommended)
  • Incompatible Detergents
    • Nonidet P-40 (which can no longer be purchased; Sigma is substituting CA- Igepal 630)
    • Triton® X-100 (or any derivative)
    • Igepal/PEG (any derivative)
    • Brij®-35 (or any derivative)
    • Tween®-20 OTG
    • CHAPSO
    • Type NP40/NP40 alternative

In-gel Digestion

  • Compatible Detergents
    • SDS (up to 2%)
    • CHAPS (up to 4%) (although not recommended)
    • Nonidet P-40 (up to 1%); which can no longer be purchased; Sigma is  substituting CA-Igepal 630 (not compatible)
  • Incompatible Detergents
    • Triton® X-100 (or any derivative)
    • Tween®-20
    • Igepal/PEG (any derivative)
    •  Brij®-35 (or any derivative)
    • OTG
    • CHAPSO
    • Type NP40/NP40 alternative

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

    • 1) N-octyl-β-glucopyranoside 
    • 2) PPS Silent Surfactant (acid-cleavable detergent)
    • 3) Protea Biosciences (anionic, zwitterionic, or cationic acid labile detergents)
    • 4) Big CHAP deoxy  (merck) h
    • 5) ASB series  (EMD chemicals) 
    • 6) sodium deoxycholate 

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.

Last modified: Feb 09, 2017