Dear Ekhlas,

When you analyze peptides from an in-gel digested protein, you will identify not only the stained protein that you excised but also all the background that is all over the gel area, such as skin keratins, enzyme that is used to digest the protein, laboratory, surface contaminants such as Casein (used as a blocking agent in Western blots; the concentration of usual blocking agents such as Casein [powdered milk] or BSA is around 5% in w/v ) ..

I am probably missing some IDs that you may be seeing and which may be originating eg., from your purification system (eg, if you use IgG, the light chain is also around 25kDa).  The top contaminants are usually keratins and you can reduce keratin contaminants by wearing powder free gloves and rinsing all the glassware with:

80% acetic acid, followed by water, followed by methanol, followed by water.

Use dedicated glassware for in-gel digestion and do not share the glassware with Western blotting experiments.

This procedure will remove all the background proteins from your gel surface and equipment, that you can consider contaminants.

All the best,

Hediye.

Presumably you know the organism from which the protein originates, so you can filter your results on that basis if genomic information is available.

Greater purity of the protein band can be obtained by 2-D electrophoresis.

I  will suggest  the use of 2D-DIGE and also the DeCyder software afterwards where the Differential In-Gel Analysis (DIA) module in DeCyder software is used to perform protein spot detection and quantification based on the area, slope, volume, peak height of the proteins. After detection, the artifacts will be removed using the exclude filter and the filtration is made to eliminate the spots that do not indicate the presence of a protein in a gel.The pick-list can be generated afterwards and the spots considered for picking could be chosen based on the protein volume ratio followed by digestion and mass spec. identification against the database.

Hi Ekhlas,

Label-free quantitation, as suggested by Andreas  above, will give you a sense of the intensity of the peptides identified and therefore, the amount of proteins identified..

MaxQuant is a free software and can provide such information (label-free quantitation).

I also agree that having the image of your gel may help us be more helpful to you, in efforts to answer your questions..

I am not sure that 2D gel electrophoresis is going to overcome your problems..These days, the mass spectrometers are able to deal with mixture of more than one protein in a sample, very successfully.  2D gel electrophoresis is yet another step and investment you have to account for.

Best,

Hediye.

Dear Dr. Hediye,

thank you for your answer.

the proteins that we have found were not related to contamination. Example of these proteins as follow:

- 14-3-3 protein zeta/delta 

- Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide

- 14-3-3 protein zeta/delta (Fragment) OS=Homo sapiens GN=YWHAZ PE=2 SV=1

- Heat shock protein beta-1 OS=Homo sapiens GN=HSPB1 PE=1 SV=2

thank you again 

Dear Dr. Adam, Dr. Pavel and Dr. Muinat,

Yes, I have tried with 2 D page techniques but the points seem to be same, the result was with many proteins.  

Thanks all for your suggestions.

Dear Dr. Andreas

Protein extract was obtained from formalin fixed tissues by standard protocol (Addis et al., 2009), the extraction buffer (EB) containing 20%w/v ratio of 20 mm Tris-HCl pH 8.8, 2% SDS, and 200 mm Dithiothreitol (DTT). 

Kindly see the attached file.

thank you for your suggestions

I am grateful for all your answers.

Many thanks for all for these useful information and suggestions

Hi Ekhlas,

I also recommend you try the free software MaxQuant as Hediye did. It will provide the intensity of each peptide and protein identified, which could be use as an reference to pick out the most abundant  proteins in your list.

Also, MaxQuant will mark the common contaminate proteins, such as Keratin and BSA mentioned above. It can help you to rule out some proteins.

Best,

Zeyang 

Dear Ekhlas,

It looks like the result is not as complicated as it seems:

1. Protein identified as "14-3-3 protein zeta/delta" is similar or same as "Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide".  "14-3-3 protein zeta/delta (Fragment) OS=Homo sapiens GN=YWHAZ PE=2 SV=1" is also the same protein, missing some amino acids, probably deposited in the Database used to search your raw, mass spec data.  Entry of truncated or partial protein sequences are common problems with Databases.  If you align the amino acid sequence of all the three above, you may find that they are one and the same protein. 

Inspection of the peptide identifications should also reveal the same conclusion above.  Just look at the list of peptides that identify the three proteins listed.

2. Heat shock protein beta-1 has a calculated Mr of ~22.8 kDa.  This is a second unique protein you have as identification.  The 14-3-3 zeta/delta has a calculated Mr of 28.2kDa. 

3. It is very possible that the ~25kDa gel bound unknown protein contains a mixture of 14-3-3 as a fragment of the full length protein and Heat scock protien beta-1.

4. The best way to confirm 1-4 above is, again, to inspect the peptide identifications and how they to the published amino acid sequences of the proteins identified.

Best,

Hediye.