Mass Quanitification of Phosphorylated Proteins in Human Cells

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Will
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Mass Quanitification of Phosphorylated Proteins in Human Cells

Postby Will » Thu May 16, 2013 8:03 am

I am planning a phospho-proteomics experiment and I need to know the weight of phosphorylated proteins per dry mass of human cell.

tsbatth
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Postby tsbatth » Thu May 16, 2013 8:26 am

Is that a question or a statement :p ? Perhaps protein/peptide quantification followed by TiO2 enrichment and perform quantification on that as well ? Getting dry weight might be difficult (% is really small I believe) but perhaps somebody has some experience in this.

Will
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Postby Will » Thu May 16, 2013 8:44 am

After a tryptic digestion of 200ug of human or yeast cell lysate, we typically recover ~100ug of tryptic peptides. After IMAC enrichment of 100ug of peptides, what is a ballpark figure for mass (ug) of phosphorylated peptides recovered? for yeast? for human?

ranio
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Postby ranio » Thu May 16, 2013 6:31 pm

Will wrote:After a tryptic digestion of 200ug of human or yeast cell lysate, we typically recover ~100ug of tryptic peptides. After IMAC enrichment of 100ug of peptides, what is a ballpark figure for mass (ug) of phosphorylated peptides recovered? for yeast? for human?

1 µg or less(0.1 µg)? I haven't measured this, due to the low abundance of phospho-proteins in vivo, it's really hard to evaluate the quantity of phospho-peptides. Looking forward others to sharing the experience.

tsbatth
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Postby tsbatth » Fri May 17, 2013 2:07 am

ranio wrote:1 µg or less(0.1 µg)? I haven't measured this, due to the low abundance of phospho-proteins in vivo, it's really hard to evaluate the quantity of phospho-peptides. Looking forward others to sharing the experience.


From what I remember, and I'm probably wrong, in human cell lines, the % phosphorylation is under 1%, but when you stimulate with something I think it goes from 1-6%. With Yeast I'm guessing the highest % would be in the growth phase but I'm not sure . I know for human cell lines normally 10-30 MG of protein is digested, fractionated, and enriched for phospho (enrichment % is normally 70-90 % if done thoroughly) before seeing good results.

ranio
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Postby ranio » Wed May 22, 2013 12:25 am

Yep, in my experience, the abundance of phosphorylation is evaluated nearly 0.1% in not-stimulated mouse liver samples, 10-15mg of proteins is digested and fractionated with SCX, after enrichment with TiO2, 5,000 phospho-peptides could be detected approximately.
tsbatth wrote:From what I remember, and I'm probably wrong, in human cell lines, the % phosphorylation is under 1%, but when you stimulate with something I think it goes from 1-6%. With Yeast I'm guessing the highest % would be in the growth phase but I'm not sure . I know for human cell lines normally 10-30 MG of protein is digested, fractionated, and enriched for phospho (enrichment % is normally 70-90 % if done thoroughly) before seeing good results.


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