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 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.

Keywords: Fusion; SPS; Tandem mass tags; multiplexing; pancreatic cancer; pancreatitis; phosphopeptide enrichment; synchronous precursor selection.

Figure 1. Experimental overview

Samples for TMT analysis were prepared as illustrated above. PaSC were harvested following 12 hr serum starvation and subsequent 12 hr treatment with either nicotine or α-bungarotoxin. Cells were harvested and lysed so that proteins could be extracted and prepared for TMT-based mass spectrometry analysis. The additional step of TiO2 enrichment was performed for phosphopeptide analysis. Control samples were labeled with 126, 127N, and 127C; nicotine-treated samples were labeled with 128N, 128C, and 129N, while α-bungarotoxin-treated samples were labeled with 129C, 130N, and 130C.

Figure 2. Western blotting analysis revealed nAChR protein expression in PaSC

The expression levels of the nicotine-binding α-subunits of nAChR in PaSC were assessed via western blotting. The neuroblastoma-like SH-SY5Y cell line was used for comparison.

Figure 3. Volcano plots illustrate statistically significant protein abundance differences in cells treated with nicotine or α-bungarotoxin

These volcano plots display the –log10 p-value versus the log2 of the relative protein abundance of A) average nicotine or B) average α-bungarotoxin to average control. Blue circles represent proteins with changes in abundance of greater than 1.5 fold and p-value >0.01.

Figure 4. Representative proteins displaying altered expression due to nicotine treatment

Plotted are relative TMT signal-to-noise levels for the selected proteins across the three controls, three nicotine-treated, and three α-bungarotoxin-treated cell cultures. The proteins highlighted include: A) alpha-2-macroglobulin (A2M), B) tweety homolog 3 (TTYH3), C) CD63, D), lysosomal protein transmembrane 4 alpha (LAPTM4A), E) lysosomal sialidase (NEU1), and G) actin (ACTB). Ctr, control; nic, nicotine; bxt, α-bungarotoxin.

Figure 5

Volcano plots illustrate statistically significant phosphopeptide level differences in cells treated with nicotine or α-bungarotoxin. These volcano plots display the –log10 p-value versus the log2 of the relative peptide phosphorylation level differences of A) average nicotine or B) average α-bungarotoxin to average control. Blue circle represent proteins with phosphorylated peptides which show changes in phosphorylation levels of greater than 1.5 fold and p-value >0.01. Closed blue circles indicate those with the highest fold changes.

Figure 6. Representative peptides displaying altered peptide phosphorylation levels due to nicotine or α-bungarotoxin treatment

Plotted are the resulting TMT signal-to-noise level for the select phosphorylated peptides across the three controls, three nicotine-treated and three α-bungarotoxin-treated cell cultures. The phosphorylated peptides include: A) T146 from HIT1H1E, B) S483 from SLC16A1, C) S573 from RRBP1, D) S1219 from SRRM2, E) S2426 from SRRM2, and F) S244 from ZBTB38. In addition, the dotted line indicates the protein expression level corresponding to the graphed peptide. The underlined amino acid represents the phosphorylation site. CTR, control; NIC, nicotine; BTX, α-bungarotoxin.