Hi Hazem,

If, based on the limited information provided, I understand your question correctly, I think the answer is undersampling.

How complex is the proteomic sample you're referring two?

What kind of fractionation/separation is done prior to MS analysis?

How abundant is your protein of interest (i.e. based on how many peptides was it identified originally)?

And are you talking about two (technical) replicates of the sample?

Many proteomic samples are so complex that fractionation/separation still results in large numbers of peptides co-eluting at certain points in your analysis. As a result, the MS cannot analyze (fragment) all peptides at these instances. If the collection of peptides it can analyze differs between two runs, it might be that a certain peptide (and thus protein) is missed during one analysis, but not in the other.

Hope this helps!

 Many Thanks Eef Dirksen

My samples are bovine sperm and I am interesting in specific protein in sperm, and I separated protein by Gel BioRad, then extract and digest by Trypsin, I follow this reference http://www.nature.com/nprot/journal/v1/n6/full/nprot.2006.468.html

So another samples  I digested lysate protein directly by trpsin with out gel then run , so I have replicated for each three biological replications each fractions  in MS,I found some protein , moreover if I repeated everything same I could not find same protein, despite  I have used same conditions and ways (lysate, volume, digestion)

so some proteins even now I could not find them, although I found them in other techniques 

Hi,

It is not strange that you're not finding a protein back in a direct in solution digest, compared to running a gel first and doing in gel digestion. Nor is it surprising that you're not finding the protein in replicate analyses.

Have you considered setting up a targeted method for your protein of interest?

Good luck! 

Hi Dirksen, Yes sure, I run my samples and I cut gel in half, then I stained one  half with coomassie dye  and another  I transferred to membrane then develop western blot and switch with another half gel  an cut slices of gel to digest, I found about thousands different types of protein but not mine.