Mass Spectrometry Sample Preparation and Submission

Columbia Users: In-house samples for analysis by mass spectrometry may be brought to room 16-424 in the P&S building during normal business hours. The tubes should be clearly labeled with your name, sample name, and date. At the time of submission, you will be asked for your e-mail address, PI, and a valid Columbia account number to which the work will be charged.  To save time, fill out and print the submission form.

Outside Users:  Shipping and payment instructions may be found here.

Gel samples for Protein Identification: Extreme care should be taken to use clean glassware and high purity reagents to minimize keratin contamination. Always wear gloves when handling gels and use a new blade when cutting the bands. Cut bands may be stored at -20 deg until they are ready to be analyzed.

Better results are obtained with tight, well-focused bands in thin gels than with diffuse bands in thick gels. If necessary, several narrow bands can be combined for a single analysis.

Western blotting: Western blotting and similar blotting procedures are far more sensitive than mass spectrometry and are specific for a particular protein; it is not reasonable to assume that if a protein can be detected on a Western blot that it will be identified by mass spectrometry in the corresponding gel piece. Mass spectrometry will detect the most abundant proteins in the band first, so several steps of purification are recommended to remove uninteresting proteins before analysis by mass spectrometry. The protein to be analyzed should be detectable by staining.

Staining: Any type of Coomassie stain may be used; bands visible by Coomassie generally contain enough protein to be successfully identified. Fluorescent stains such as Sypro Ruby are also compatible with mass spectrometry and may be used, but they pose practical problems in visualizing and cutting the band.

Identification of silver stained bands has met with some success (~25-40%), though less overall than with Coomassie-stained bands. If silver must be used, the silver stain must be compatible with mass spectrometry. Commercial silver stains are available which specifically state that they are mass spec compatible. Homemade silver stains tend to produce fewer artifacts in the spectrum and there are numerous protocols for MS-compatible stains; the one below is typical:

Silver Staining for Acrylamide Gels

1. After electrophoresis, fix gels in methanol/acetic acid/H2O (50:5:45)
   250ml methanol, 25ml acetic acid, 225 ml H2O/500ml
   
   
2. Wash with H2O, 3 changes for 2 min/2min/30-60 min
   
   
3. Incubate in sensitizing solution (0.02% sodium thiosulfate) for 2 min
   Make fresh 0.2%  10X stock --- 1g/500ml
   
   
4. Wash with 2 changes of H2O, 10 sec each.
   
   
5. Incubate with chilled 0.1% silver nitrate for 30 min
   100mg/100ml ----- 500mg/500ml
   
   
6. Wash with 2 changes of H2O, 30 sec each.
   
   
7. Incubate in developing solution, 0.04% formaldehyde in 2% sodium carbonate until desired degree of staining.
   15g sodium carbonate, 250 µl 37% w/w formaldehyde/ 500ml
   
   
8. Terminate staining and store gels in 1% acetic acid
   10ml/1000ml

Mass Analysis of Intact Proteins: Proteins are generally analyzed by MALDI-TOF which can measure masses up to 150kD, although the higher the mass, the higher the protein concentration must be to produce a good spectrum. Proteins should be submitted in solution, 10-20 µl of at least 1mg/mL up to 30kD, 2-5 mg/mL 30-70kD, and >5mg/mL for high molecular weight proteins. Low salt concentration (<50mM) will not interfere, but higher salt and non-ionic detergents will compromise the spectrum.

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