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Protein Identification By Mass Spectrometry

 

 

 

We offer high sensitivity protein identification from gel spots or bands using the latest technologies in mass spectrometry. In each run, four sensitivity standards in the amount of 1 femtomole are included. Our sensitivity is 1 femtomole.

Sample type: well resolved 1D or 2D gel spots containing only 1 protein species

Guidelines for Preparing and Shipping Samples for Protein Identification


Reduction, Alkylation, Gel Washing
& In-gel Trypsin Digestion
 
Protein identification by mass spectrometry workflow
Desalting w/ Zip-tip C18 (Millipore)
Spotting on MALDI plate
MALDI-TOF
MALDI-TOF/TOF

Database Search for Protein ID

 

1. Gel treatment: 2D gel spots are washed multiple times to remove staining dye and other inhibitory chemicals. Gel spots are then dried to absorb maximum volume of digestion buffer.

2. In-gel trypsin digestion: Dried 2D gel spots are rehydrated in digestion buffer containing sequencing grade modified trypsin. Proteins are digested in-gel at 37°C.

3. Peptide extraction: Digested peptides are extracted from gel with TFA extraction buffer and shaking.

4. Desalting: The digested tryptic peptides are desalted using C-18 Zip-tips (Millipore).

5. Spotting: The desalted peptides are mixed with CHCA matrix (alpha-cyano-4-hydroxycinnamic acid) and spotted into wells of a MALDI plate.

6. MALDI-TOF: Mass spectra (MS) of the peptides in each sample are obtained using an Applied Biosystems Proteomics Analyzer.

7. MALDI-TOF/TOF: Ten to twenty of the most abundant peptides in each sample are further subjected to fragmentation and tandem mass spectrometry (MS/MS) analysis.

8. Database search: Protein identification is based on peptide fingerprint mass mapping (using MS spectra) and peptide fragmentation mapping (using MS/MS spectra). Combined MS and MS/MS spectra are submitted for database search using GPS Explorer software equipped with the MASCOT search engine to identify proteins from primary sequence databases.

9. Data Report: Includes the top 10 highest scoring hits from the database search for each 2D gel spot, as well as a summary listing the best match for each sample (see a sample report of protein ID analysis)

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Guidelines for Preparing and Shipping Samples for Protein Identification

1-D gel band: Please keep in mind that a band from a 1D gel could contain several proteins, although it looks a single band. This can reduce the real amount of your protein of interest. Low quantity and high complexity of samples will greatly reduce the chance of success for high confident identification. Please follow the tips below:

1. Protein amount. Our general rule of thumb is that if there is enough protein to be strongly stained with Coomassie G-250, there should be enough for Mass Spectrometry analysis. We prefer that you scale up your prep so that you have 10-20 ng of protein in the gel band. Please do not scale up by sending multiple gel pieces, as this increases gel volume and decreases the efficiency of peptide extraction from the gel. Instead, load a higher amount of protein into ONE lane of the gel.

2. Minimize gel volume. It is important to minimize the gel volume and maximize protein concentration. As such, we recommend you cut out only the area with your protein of interest (1x1x2mm size) and exclude as much unstained gel as possible.

3. Silver stained gel band should be compatible with Mass Spectrometry. You need to make sure your silver staining solution does not contain aldehyde that cross-links your proteins to the gel matrix, resulting in low extraction efficiency. Non-formaldehyde Silver Stain uses carbohydrazide instead of formaldehyde for reduction/development.

4. Shipping condition. When you are ready to send your sample, please add HPLC-grade water to cover the excised gel band in a 1.5ml eppendorf tube, or 96 well plate. Seal the Eppendorf tubes with Parafilm. Put the Eppendorf tubes into 50 mL conical tubes, then pack the 50 mL tube with Kimwipes to prevent the Eppendorfs from being crushed or jostled. The samples can be shipped on ice pack (no need to send on dry ice) to the following address:

Proteomics Service
Applied Biomics, Inc.
23785 Cabot Blvd, Suite 312
Hayward, CA 94545
USA


5. Order form. Fill out the order form . The completed form can be included in the package, or faxed or emailed to us.