Phosphate buffer can be a bad choice for freezing delicate proteins because it undergoes pH change during freezing/thawing:

http://www.ncbi.nlm.nih.gov/pubmed/11368330

Rapid freezing and thawing is important when using phosphate buffer.

I was wondering why you chose phosphate buffer. What is your protein sample? I recommend you that do not use stabilizers and change your buffer. 

Dear Adam,

Thanks for your reply,actually i freezed my sample directly from liquid nitrogen to -80 c