Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells.
Smoking is a risk factor in pancreatic disease; however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12 h in triplicate and compared alterations in protein expression and phosphorylation levels to mock-treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8100 proteins were quantified across all nine samples, of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease, and lysosomal proteins. In addition, we measured differences for ∼16 000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease.
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Joao A
JA
Paulo
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Aleksandr
A
Gaun
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Steven P
SP
Gygi
D002038:Bungarotoxins / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009538:Nicotine / Q000494:pharmacology*; D058954:Pancreatic Stellate Cells / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D002038:Bungarotoxins; D008024:Ligands; D010750:Phosphoproteins; D020543:Proteome; D011978:Receptors, Nicotinic; D009538:Nicotine
Fusion; SPS; Tandem mass tags; multiplexing; pancreatic cancer; pancreatitis; phosphopeptide enrichment; synchronous precursor selection