MassIVE MSV000091198

Partial Public PXD039843

Quantitative phosphoproteomics to define cAMP compartments in Human Airway Smooth Muscle cells

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Description

The hypothesis driving this present study is that cAMP signaling occurs in specific compartments defined primarily by the expression of distinct adenylyl cyclase (AC) isoforms. By using a direct activator of AC activity to stimulate cAMP in all pools of the cell but manipulating just one pool by overexpressing one isoform of AC at a time, we can perform subtractive analysis to determine the activated signals in a single cAMP compartment. We performed quantitative phosphoproteomics using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to identify all phosphorylated proteins in Human Airway Smooth Muscle (HASM) cells resulting from a short stimulus of AC activity. By separately overexpressing two different isoforms of AC, AC2 and AC6, that are localized in separate plasma membrane microdomains and then comparing these data to that from control cells (with their native expression of these two AC isoforms in addition to others), we can define the phosphoproteomic profile of cAMP signal emanating from each of these two compartments. Protocol: Human Airway Smooth Muscle (HASM) cells were grown in media containing 13C labeled lysine and arginine (heavy media) or in normal (light) media for 15 days. Cells were then incubated with recombinant adenoviruses expressing either lacZ, AC2, and AC6 for 24 hr. Heavy cultures of HASM cells were stimulated with 1 microM forskolin for 10 min. Lysates of treated heavy cells were mixed with lysates from vehicle-treated light cells. This created SILAC pairs for forskolin stimulation in each of the three cell conditions (lacZ, AC2, or AC6). Proteins were digested, enriched for phosphopeptides by TiO2 column, and analyzed by LC/MS/MS by the proteomics core facility at Johns Hopkins School of Medicine. The TiO2-enriched samples were run. Mass spec measured the relative abundance of a phosphopeptide when a heavy and light peptide pair (a SILAC pair) was detected in the samples. The quantitative difference represents the change in abundance of that peptide caused by the forskolin treatment. Peptide sequencing, database search, protein identification, and analysis of post-translational modifications of peptides was performed by the core. 7,000 to 10,000 confirmed phosphopeptides were detected in the enriched samples, corresponding to 2,100-2,500 unique proteins. [doi:10.25345/C5HH6CG3W] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: adenylyl cyclase ; Human Airway Smooth Muscle ; cAMP compartments

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Principal Investigators:
(in alphabetical order) 		Rennolds Ostrom, Ph.D., Chapman University School of Pharmacy, USA
Submitting User: roosan
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