Nicotine-induced protein expression profiling reveals mutually altered proteins across four human cell lines

Abstract

Mass spectrometry-based proteomic strategies can profile the expression level of proteins in response to external stimuli. Nicotine affects diverse cellular pathways, however, the nicotine-induced alterations on the global proteome across human cell lines have not been fully elucidated. We measured perturbations in protein levels resulting from nicotine treatment in four cell lines-HEK, HeLa, PaSC, and SH-SY5Y-in a single experiment using tandem mass tags (TMT10-plex) and high-resolution mass spectrometry. We quantified 8590 proteins across all cell lines. Of these, nicotine increased the abundance of 31 proteins 1.5-fold or greater in all cell lines. Likewise, considering proteins with altered levels in at least three of the four cell lines, 64 were up-regulated, while one was down-regulated. Gene ontology analysis revealed that ∼40% of these proteins were membrane bound, and functioned in transmembrane signaling and receptor activity. We highlighted proteins, including APP, APLP2, LAPTM4B, and NCOA4, which were dysregulated by nicotine in all cell lines investigated and may have implications in downstream signaling pathways, particularly autophagy. Using the outlined methodology, studies in additional (including primary) cell lines will provide further evidence that alterations in the levels of these proteins are indeed a general response to nicotine and thereby merit further investigation.

Keywords: Cell biology; Nicotine; Orbitrap fusion; Pancreas; SPS-MS3; TMT.

Figure 1

Experimental overview of the SPS-MS³ strategy. Four cell types (PaSC—in duplicate, HEK293, HeLa, and SH-SY5Y) were propagated and designated cultures were mock or nicotine treated. Proteins were extracted and then digested with Lys-C and trypsin. The resulting peptides were labeled with TMT, pooled, and fractionated via basic pH reversed-phase high performance liquid chromatography (BPRP-HPLC) prior to MS3 analysis on an Orbitrap Fusion mass spectrometer.

Figure 2

Total of 8590 proteins were quantified by using the SPS-MS3 strategy. (A) Heat map and associated dendrogram of samples analyzed in the TMT10-plex experiment. Data were normalized to the average untreated PaSC samples. (B) Spearman correlation (ρ) matrix of protein abundance for all ten samples. Highlighted in red and blue are the correlations of the replicates for PaSC and PaSC+nicotine, respectively, both of which demonstrated ρ>0.9. nic, nicotine.

Figure 3

Venn diagrams of proteins with altered abundance following nicotine treatment. The Venn diagrams display the protein overlap for (A) 402 non-redundant proteins that exhibited an increase (≥1.5-fold) in abundance, and (B) the 32 that exhibited a decrease (≥1.5-fold) in abundance when the cell lines were treated with nicotine for 24 h.

Figure 4

Proteins (n = 31) demonstrating a significant increase in abundance following nicotine treatment in all four cell lines. Heat map scale is the percentage of the total abundance of a protein across all ten channels.

Figure 5

Bar graphs of proteins differentially expressed with statistical significance across cell lines. (A–H) Plots illustrate examples of proteins that demonstrate abundance greater than 1.5-fold in at least three of the four cell types investigated. (I) Actin is plotted as a control and does not show significant alteration in abundance in any cell line following nicotine treatment.

Figure 6

Network of 65 proteins that demonstrate differences in abundance (fold change ≥1.5). Line thickness of the edges indicates the strength of data supporting the interactions. Analysis was performed by using String [60] and evidence connecting nodes included experiments, databases, co-expression, neighborhood, gene fusion and co-occurrence.