title: Sensitive targeted quantification of ERK phosphorylation dynamics and stoichiometry in human cells without affinity enrichment.
abstract: Targeted mass spectrometry is a promising technology for site-specific quantification of posttranslational modifications. However, a major constraint is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents for enrichment. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometry using a sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection, and multiplexing (PRISM). PRISM provides effective enrichment of target peptides into a given fraction from complex mixture, followed by selected reaction monitoring quantification. Direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) was demonstrated from as little as 25 μg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided ∼10-fold higher signal intensities, presumably due to the better peptide recovery of PRISM. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of epidermal growth factor at both the peak activation (10 min) and steady state (2 h). The maximal ERK activation was observed with 0.3 and 3 ng/mL doses for 10 min and 2 h time points, respectively. The dose-response profiles of individual phosphorylated isoforms showed that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99354, United States.
authors.forename: Tujin
authors.initials: T
authors.lastname: Shi
authors.forename: Yuqian
authors.initials: Y
authors.lastname: Gao
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Gaffrey
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Fillmore
authors.forename: William B
authors.initials: WB
authors.lastname: Chrisler
authors.forename: Marina A
authors.initials: MA
authors.lastname: Gritsenko
authors.forename: Chaochao
authors.initials: C
authors.lastname: Wu
authors.forename: Jintang
authors.initials: J
authors.lastname: He
authors.forename: Kent J
authors.initials: KJ
authors.lastname: Bloodsworth
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: H Steven
authors.initials: HS
authors.lastname: Wiley
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D001940:Breast / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods*; D004847:Epithelial Cells / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D048049:Extracellular Signal-Regulated MAP Kinases