A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell–Cell Induced Protein Secretion

Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell–cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, “physiological” forebrain neurons. “Nonphysiological” cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins

Characterization of the HeLa Δ<i>SGPL1</i> cell lines.

<p>(A) HeLa wildtype (<b>H</b>, Lane 1) and HeLa Δ<i>SGPL1</i> (<b>Δ</b>,Lane 2) cells were labeled with 6 μM pacSph for 6 h. Lipids were extracted and subjected to click reaction with fluorogenic fluorescein, separated by TLC and excited with blue light. Lipids were identified based on comparison to the respective lipid standards (see Fig 2C). Both samples were also subjected to alkaline hydrolysis (Lane 3 and 4). (B) Quantification of the fluorescence intensities of Fig 2C. Mean ± SD (n = 3) are shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s019" target="_blank">S2 Table</a>). Each timepoint is standardized to 100% of lipids quantified. Data are fitted by Local Polynomial Regression Fitting (LOESS, degree = 2) as curves. (C) HeLa Δ<i>SGPL1</i> cells were labeled for the time points indicated and treated as in (A). As reference, 50 pmol of each clickable Cer, HexCer, PC and SM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s004" target="_blank">S3 Fig</a>) was loaded as standard.</p

Characterization of the MEF <i>Sgpl1</i>^+/+ and <i>Sgpl1</i>^-/- cell lines.

<p>(A) <i>Sgpl1</i>^+/+ and <i>Sgpl1</i>^-/- cells were metabolically labeled with 6 μM pacSph for 4 h. Lipids were extracted and subjected to click reaction with fluorogenic coumarin azide, separated by TLC and exited with UV light. Lipids were identified via clickable standards (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s004" target="_blank">S3 Fig</a>). Three replicates are shown for every cell line. (B) Quantification of the fluorescence intensities of Fig 1A. Values are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s018" target="_blank">S1 Table</a>. Mean ± SD are shown (n = 3). (C) Samples were treated as above, but were also subjected to alkaline hydrolysis (saponified) or mock treatment. (D) Membrane Lipidome. Lipid class profile of <i>Sgpl1</i>^+/+ and <i>Sgpl1</i>^-/- cells. Lipid classes are standardized to all lipids measured excluding storage lipids (TAG and CE). GP O- lipids are contained in the sum of each class (e.g. PE O- in PE) and may show overlap with diacyl species containing odd-numbered fatty acids. PE Plasmalogens (PE P-) are displayed as a separate class. A Welch Two Sample t-test was used to estimate the P values: *P < 0.05; **P < 0.01; *** P < 0.001. Error bars correspond to standard deviation (n = 6).</p

Lipidomics analysis of the HeLa and HeLa Δ<i>SGPL1</i> cell lines.

<p>(A) Class profile, standardized to all lipids measured without storage lipids (TAG and CE). GP O- lipids are contained in the sum of each class (e.g. PE O- in PE) and may show overlap with diacyl species containing odd-numbered fatty acids. PE P-lipids are displayed as a separate class. (B) Sphingoid bases and sphingoid base 1-phosphates standardized to total phosphate (fmol/nmol P_i). Data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s020" target="_blank">S3 Table</a>. A Welch Two Sample t-test was used to estimate the P values: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars correspond to standard deviation (n = 3).</p

Fluorescent labeling of pacSph metabolites to study protein lipid interaction.

<p>Flag-tagged STARD7 (A) and p24 (B) were expressed in HeLa and HeLa Δ<i>SGPL1</i> cell lines. Cells were metabolic labeled with 5 μM pacSph (STARD7) or 0.5 μM pacSph with or without sphingosine (p24) for 7 h and then UV irradiated to cross-link pacSph metabolites to nearby protein. Protein lysates were subjected to click reaction with Alexa647 azide (shown in red) and the ectopically expressed proteins were immunoprecipitated. After SDS-PAGE and immunoblot with fluorescently labeled secondary antibodies (shown in green), lipid and protein signals were detected in separate channels. Lys, Lysate, IN, input of immunoprecipitation, IP, immunoprecipitated material.</p

Proteomic analysis of the HeLa and HeLa <i>ΔSGPL1</i> cell lines.

<p>Differential expression of proteins in HeLa and HeLa Δ<i>SGPL1</i> cell lines. <b>(A and C)</b> Proteins only found in one of the cell lines. Protein LFQ (label free quantification) intensities of HeLa (A) or HeLa Δ<i>SGPL1</i> (C) cells were used to calculated T-test p-values. A list of proteins is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s022" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s023" target="_blank">S6</a> Tables. <b>(B)</b> Volcano plot of differential expression of proteins. Difference of log transformed LFQ (label free quantification) intensities of HeLa cells and HeLa Δ<i>SGPL1</i> cells (Δ<i>SGPL1</i> –HeLa). Statistical analysis was carried out with Perseus software. Significance Values are based on permutation based FDR analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.ref044" target="_blank">44</a>]: Proteins scoring p < 0.05 are shown in green, p < 0.01 in red. A list of proteins is provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s021" target="_blank">S4 Table</a>.</p

Rescue of HeLa Δ<i>SGPL1</i> cells by exogenous Flag-SGPL1.

<p>HeLa wildtype, HeLa Δ<i>SGPL1</i> and HeLa Δ<i>SGPL1</i> transfected with Flag-tagged SGPL1 were labeled with 6 μM pacSph for 4 h. <b>Upper Panel:</b> Proteins precipitated during the lipid extraction were solubilized, analyzed by SDS-PAGE and immunoblotting, using anti-FLAG antibodies (red). Detection of endogenous calnexin with anti-calnexin antibody (CNX, green) was used as a loading control. <b>Lower Panel:</b> Extracted lipids were subjected to click reaction with fluorogenic coumarin azide, separated by TLC and exited with UV light. Lipids were identified via with alkyne lipid standards (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.s004" target="_blank">S3 Fig</a>).</p

Oligonucleotides inserted into the BbsI site of pSpCas9(BB)-2A-GFP.

<p>sgRNA sequences are indicated in bold and preceded by a G nucleotide to improve transcription [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153009#pone.0153009.ref029" target="_blank">29</a>].</p