Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells

Abstract

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 12hr in triplicate and compared alterations in protein expression and phosphorylation levels to mock treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8,100 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.

USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites

“…Fragment ions generated by HCD (normalized collision energy was set to 27%) were acquired in the Orbitrap (70k resolution, AGC of 1 × 10 6 , 200 ms of maximum injection time and 1 m/z isolation window). RAW files obtained were processed using an in-house software pipeline based on Sequest 27,28 . Signal to noise ratio of the reporter ions was used to calculate the abundance of each linkage across all samples.…”

Multiplexed Isobaric Tag‐Based Profiling of Seven Murine Tissues Following In Vivo Nicotine Treatment Using a Minimalistic Proteomics Strategy

“…We aimed to perform a large-scale analysis of mouse tissue while efficiently processing samples. Previously, protein level TMT-based mass spectrometry protocols [6, 16, 17] recommended 50–100 μg of peptide per condition, however, only one-fifth to one-tenth of the sample is typically analyzed by the mass spectrometry, the remaining is either discarded or used for re-analysis in the event that errors occurred during the initial analysis. Here, we labeled only one-fifth of the typical starting material (20 μg), and thus use the proportional amount of the labeling reagent.…”

“…Likewise, Serinc3 (serine incorporator 3) is involved in sphingolipid metabolic process [21] . Serinc3 was also up-regulated in nicotine-treated cells in various cell lines including, pancreatic stellate cells [6] , HEK293 [22] , HeLa [22] , SH-SY5Y [22] , HPNE [23] and Panc1 [23] cells. Bpifa2 (BPI fold A2) lipopolysaccharide binding protein participates in lipid transport [24] , and shows a significant increase in abundance in five tissues: lung, spleen, kidney, liver, and heart.…”

“…Growing evidence has linked nicotine to the disruption of cellular metabolic processes and its potential to be genotoxic and tumor-promoting [4] . Nicotine has been shown to induce cell proliferation and invasion in multiple lung and breast cancer lines [5] , and alter the phosphorylation states of proteins in pancreatic stellate cells [6] . Previous studies link nicotine and diseases, but none have explored the proteome-wide effects on a mouse model system as examined here.…”

Isobaric Tag‐Based Protein Profiling of a Nicotine‐Treated Alpha7 Nicotinic Receptor‐Null Human Haploid Cell Line

“…Growing evidence has linked nicotine to the disruption of cellular metabolic processes and its potential to be genotoxic and tumor-promoting [5] . Nicotine has been shown to induce cell proliferation and invasion in multiple lung and breast cancer lines [6] , and to alter the phosphorylation states of proteins in pancreatic stellate cells [7] . Various cancers and chronic diseases have been linked to nicotine, yet the mechanisms thereof remain poorly understood [8] .…”