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Urgent help - 2DE-MALDI

Urgent help - 2DE-MALDI - Proteomics Forum

Urgent help - 2DE-MALDI - Post Questions and Discuss Proteomics, Proteomic Bioinformatics, Proteomic Techniques such as 2-D, Mass Spec etc.


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Old 06-05-2008, 02:11 PM
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Default Urgent help - 2DE-MALDI



I have recently undertaken my first proteomics experiment (protein extraction from cells, 2DE, exicised spots, trypsin digestion, MALDI-TOF MS) but have not had much success and I was wondering if anyone could help me. I struggled to get any identifications because I seem to have quite a lot of artefactual peaks that appear across a number of samples. Although I used a laminar flow hood, wore gloves, a lab coat and covered my hair, I seem to have a number of contaminants. Some of them appear to be kertains and trypsin peaks, but I cannot identify the others. I was wondering if you had any suggestions as to what they might be? They are at m/z 1149, 1251, 1267, 1295, 1412, 1759 and 2186. They are towering above my keratin/trypsin peaks and peaks of interest. Also, does anyone know where I might be able to find a peak list of common contaminants (from keratins, trypsin, matrix etc.)?

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Old 06-05-2008, 03:34 PM
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Default Re: Urgent help - 2DE-MALDI

Unfortunately, proteomics (mass spec) requires practice. You should invest in repeating (with changes etc), instead of cleaning up the data. There is a learning curve to performing mass spec experiments.

My suggestion is to suspect your buffers and matrix quality first.
Plastic bottles are a big no (they leak plasticides), change to glass.
Prepare fresh solutions (should last about a month).
Latex gloves are full of keratins, so either don't use them or be extremely careful about what you touch.
And of course stain your gel with fresh staining solution that is MS-compatible (I use Imperial Blue, Pierce).
If you don't want trypsin artifact--use trypsin spin columns (I use Sigma's).
I use Agilent Tech CHCA matrix--which is perfect compared to others I tried like Sigma etc.
Zip tips are great for desalting as well as Millipore ion-exchange 96-well plates.
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