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

Journal Article · Wed Dec 17 00:00:00 EST 2014 · Analytical Chemistry

DOI:https://doi.org/10.1021/ac503797x· OSTI ID:1171302

Shi, Tujin ^[1]; Gao, Yuqian ^[1]; Gaffrey, Matthew J. ^[1]; Nicora, Carrie D. ^[1]; Fillmore, Thomas L. ^[1]; Chrisler, William B. ^[1]; Gritsenko, Marina A. ^[1]; Wu, Chaochao ^[1]; He, Jintang ^[1]; Bloodsworth, Kent J. ^[1]; Zhao, Rui ^[1]; Camp II, David G. ^[1]; Liu, Tao ^[1]; Rodland, Karin D. ^[1]; Smith, Richard D. ^[1]; Wiley, H. Steven ^[1]; Qian, Weijun ^[1]

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1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

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, whereas 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.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: National Institutes of Health (NIH); USDOE
• Grant/Contract Number: AC05-76RL01830; U24-CA-16001901; DP2OD006668; P41GM103493
• OSTI ID: 1171302
• Report Number(s): PNNL-SA-103421; 48216; 400412000
• Journal Information: Analytical Chemistry, Vol. 87, Issue 2; ISSN 0003-2700
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
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