Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry.
J Proteome Res
; 17(4): 1654-1663, 2018 04 06.
Article
in En
| MEDLINE
| ID: mdl-29457462
ABSTRACT
We show that parallel reaction monitoring (PRM) can be used for exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled peptides. We have compared two different PRM approaches on a digest of a U87 cell culture, namely, direct-PRM (tryptic digest measured by PRM without any further sample preparation) and TiO2-PRM (tryptic digest enriched with TiO2 cartridges, followed by PRM measurement); these approaches are compared for the following phosphorylation sites neuroblast differentiation-associated protein (AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor (EGFR S1166-p). A reproducible percentage of phosphorylation could be determined (CV 6-13%) using direct-PRM or TiO2-PRM. In addition, we tested the approaches in a cell culture experiment in which U87 cells were deprived of serum. As a "gold standard" we included immune precipitation of EGFR followed by PRM (IP-PRM). For EGFR (S1166) and AHNAK (S5480) a statistical significant change in the percentage of phosphorylation could be observed as a result of serum deprivation; for EGFR (S1166) this change was observed for both TiO2-PRM and IP-PRM. The presented approach has the potential to multiplex and to quantify the ratio of phosphorylation in a single analysis.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Phosphorylation
/
Mass Spectrometry
Limits:
Humans
Language:
En
Journal:
J Proteome Res
Journal subject:
BIOQUIMICA
Year:
2018
Document type:
Article
Affiliation country:
Países Bajos