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Title: Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reaction monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereasmore » the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated 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.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Publication Date:
OSTI Identifier:
1171302
Report Number(s):
PNNL-SA--103421
Journal ID: ISSN 0003-2700; 48216; 400412000
Grant/Contract Number:
AC05-76RL01830; U24-CA-16001901; DP2OD006668; P41GM103493
Type:
Accepted Manuscript
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 87; Journal Issue: 2; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Research Org:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org:
National Inst. of Health (NIH); USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY Environmental Molecular Sciences Laboratory