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. 2016 Jan 4;15(1):125-43.
doi: 10.1021/acs.jproteome.5b00597. Epub 2015 Dec 15.

Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins

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Free PMC article

Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins

Judy K Cundiff et al. J Proteome Res. .
Free PMC article

Abstract

Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

Keywords: Caco-2; TMT; intestinal epithelial cells; milk proteins; quantitative proteomics.

Conflict of interest statement

Conflict of Interest Statement

The authors contributed to this work during their employment of Mead Johnson Nutrition.

Figures

Figure 1.
Figure 1.
Analysis of TMT ratio compression (from labeling to LC/MS/MS detection). (A) Schematic representation assessing TMT ratio compression. Regular Caco-2, heavy Caco-2 and yeast proteins were each prepared to obtain tryptic peptides. Regular Caco-2 peptides were then loaded at indicated stoichiometric (S.) ratios, whereas heavy Caco-2 peptides with or without yeast were loaded at equal levels, followed by TMT labeling, ERLIC prefractionation and repeated LC/MS/MS analyses. The dot frames indicate sample combinations for each 6-plex TMT channel. For simplicity, the schematic displays the interference containing both heavy Caco-2 and yeast materials. (B) Schematic representation showing the mutual interference in TMT ratios between regular and heavy Caco-2 peptides. The presence of heavy Caco-2 peptides at equal theoretical ratios (R theo) compresses the measurements of regular Caco-2 peptides from 1.05 – 1.4 Rtheo to respective experimental ratios (R˜exp) for each TMT channel. Conversely, the presence of regular Caco-2 peptides at Rtheo inflates the ratios of heavy Caco-2 peptides from Rtheo to respective (Rexp)for each TMT channel., Comparisons of the experimental medians (R˜exp) to respective Rtheo were used to calculate the compression factors (FC) and the inflation factors (Fi)for regular and heavy Caco-2 proteins, respectively. (C) Ratio distribution of regular Caco-2 proteins under HSILACi + YEASTi (C1) or HSILACi (C2) conditions. S. ratios of regular Caco-2 proteins are indicated. (D) Respective ratio distribution of equal-level heavy Caco-2 proteins under the presence of regular Caco-2 proteins at indicated S. ratios. The vertical lines in box plots indicate the median and the whisker caps indicate the lower 10th and upper 90th percentiles, respectively. HSILACi, heavy SILAC Caco-2 interference; YEASTi, yeast interference.
Figure 2.
Figure 2.
Analysis of TMT ratio compression (from cell lysis to LC/MS/MS detection). (A) Schematic representation assessing TMT ratio compression. Regular Caco-2 proteins and interfering heavy Caco-2 proteins with or without yeast were mixed at indicated stoichiometric (S.) ratios immediately after cell lysis, followed by proteomic procedures and repeated LC/MS/MS analyses. The dot frames indicate sample combinations for each 6-plex TMT channel. For simplicity, the schematic displays the interference containing both heavy Caco-2 and yeast materials. (B) Ratio distribution of regular Caco-2 proteins under HSILACi + YEASTi (B1) or HSILACi (B2) conditions. S. ratios of regular Caco-2 proteins are indicated. Ratios of regular Caco-2 proteins were normalized to median log2 zero of the equally-loaded heavy Caco-2 proteins. (C) Ratios of regular Caco-2 protein in (B) were normalized to the experimental medians (R˜exp) of heavy Caco-2 proteins (Figure 1D), instead of median log2 zero. (D) Ratio distribution of regular Caco-2 proteins with respect to relative protein abundance. Ratios of regular protein in (C) were divided into four quartiles from high to low emPAI. HSILACi, heavy SILAC Caco-2 interference; YEASTi, yeast interference. (E) Schematic representation assessing the precision of TMT ratio with respect to the number of technical replicate under independent sample preparation. Heavy Caco-2 proteins at equal levels were mixed with regular Caco-2 proteins at stoichiometric (S.) ratios of 1 to 1.1 immediately after cell lysis, followed by analogous proteomic procedures shown in Figure 2A. (F) Corresponding IQR of protein ratios at n = 1 – 12 technical replicates with single or duplicated LC/MS/MS. (G) Differences of protein ratios to be significant with respect to the number of technical replicates at significance levels α = 0.05 and β = 0.20.
Figure 3.
Figure 3.
Strategy for comparing the proteome of Caco-2 monolayer with or without human whey protein (WP) treatment over time. (A) Representative TEER trajectories of differentiating Caco-2 monolayer with (+) or without (−) successive application of WP beginning on D9. Days (D) are post seeding. Error bars represent s.e.m. (B) Schematic comparing Caco-2 proteome across six temporal stages and for two different +/−WP durations. To depict the temporal proteome of Caco-2 monolayer, cultured cells under –WP conditions (①) were harvested on D9, 11, 13, 15, 17 and 21, followed by TMT 6-plex-based proteomic procedure. To assess the effects of WP on the proteome of Caco-2 monolayer over time, cultured cells with successive +WP treatments beginning on D9 were harvested on D15 (②) or D22 (③), each grouped with respect –WP controls and followed by TMT 6-plex-based proteomic procedure. Combined results from the three studies of -WP (D9 → D21), +/−WP (D9 → D15) and +/−WP (D9 → D22) are used to depict the temporal proteome of Caco-2 monolayer with or without WP treatments. (C) Sample hierarchy including both biological replicates (n) and repeated LC/MS/MS analysis (x or y). N = 12 pairs for each of the +/−WP (D9 → D15) and the +/−WP (D9 → D22) analysis. N = 6 at each stage of D9, 11, 13, 15, 17 and 21 for the -WP (D9 → D21) analysis. Repeated LC/MS/MS were performed for each TMT sample set. (D) Venn diagram of proteins quantified from the three indicated studies. The number of proteins covered by each sample group is indicated.
Figure 4.
Figure 4.
System-wide characterization of WP-induced abundance changes of Caco-2 proteins. (A, B) Protein ratio variance between repeated LC/MS/MS analysis. (A) Profiles of protein ratio variance between repeated LC/MS/MS of 12 +/−WP sample pairs. (B) Scatter plot of protein ratio variance between repeated LC/MS/MS with respect to exponentially modified protein abundance index (emPAI) for an exemplary +/−WP sample pair. Proteins were ordered from high to low emPAI and assigned ranks where 1 corresponds to the highest emPAI. Color curves show the fraction of proteins with absolute FC variance (|ΔFC|) > 1.1 for each of the 12 sample pairs. FC, fold change. (C-D) Protein ratio variance with respect to the number of biological replicates. Ratios were from the average of two LC/MS/MS analysis. Ratio variances of +/−WP sample pair were compared between sample sizes of n+3 and n. Red, n = 3; blue, n = 6 and green, n = 9. s(n), sample size of n. (D) Scatter plots of protein ratio variance between s(n+3) and s(n) with respect to emPAI. Color curves show the fraction of proteins with |ΔFC| > 1.05. (E) Standard deviations of protein ratio at n = 9 biological replicates (“Std. Dev. (n=9)”, Supplemental Dataset 4 and 5) with respect to the number of significant peptide sequences for a linked protein; red dots and fitting line, BH p-value < 0.05; blue dots and fitting line, BH p-value ≥ 0.05; top marginal histogram, distribution of the number of unique peptide sequences; right marginal histogram, distribution of standard deviation. (F) Principal component analysis of the quantitative Caco-2 proteomes. Samples were grouped by ellipsoid according to treatment condition and duration. (G) Distribution of protein abundance changes induced by WP treatment over indicated periods. Proteins with positive or negative FCs are up- or down-regulated, respectively, under +WP conditions. Same proteins from the two different treatment durations are connected by solid lines with yellow ones highlighting significant abundance changes. The areas of dots are proportional to the standard deviations in protein abundance changes. The shade delimits −1.05 ≤ FC ≤ +1.05 or BH p-value ≤ 0.05. LTF, lactotransferrin. (H) Cross comparison of protein abundance change with respect to treatment duration. H1: mapping of 888 Caco-2 proteins (BH p < 0.05) from +/−WP (D9 → D15) dataset to +/−WP (D9 → D22) dataset. H2: mapping of 3,609 Caco-2 proteins (BH p < 0.05) from +/−WP (D9 → D22) dataset to +/−WP (D9 → D15) dataset. N = 12 for each group. To facilitate the visualization of small FCs, the color scale is limited to ±log2 1.0 with no distinction in colorimetric representation for greater magnitude of FC. In A and C: the vertical lines in box plots indicate the median and the whisker caps indicate the lower 10th and upper 90th percentiles, respectively.
Figure 5.
Figure 5.
Analysis of WP endocytosis. (A) 10% WP (light) was applied to Caco-2 cells with 13C6,15N2-L-lysine and 13C6,15N4-L-arginine-labeled proteins (heavy). Left panel: schematic of the SILAC procedure measuring light-to-heavy (L/H) protein ratios. Right panel: L/H protein ratios obtained from the SILAC setup. LTF, lactotransferrin; IGHA1, immunoglobulin heavy chain A1. (B) Caco-2 cells were cultured in standard medium for 22 days without WP (−) or with WP (+) over D9 → D22 period or on D22 for 10 minutes. Left panel, schematic of the TMT procedure assessing protein non-specific bindings. Right panel: corresponding TMT protein ratios of LTF and IGHA1 relative to -WP control. (C) Immunoblot analysis of LTF under -WP or +WP conditions over D9 → D15 or D9 → D22 periods. For samples with +WP treatment for 10 minutes, WP was applied on indicated end days. For samples with longer treatment durations, WP was applied from D9 to indicated end days.
Figure 6.
Figure 6.
Regulation of Caco-2 pathways upon WP treatment. (A1-F1) FC of Caco-2 proteins in KEGG pathways of (A1) cell cycle, (B1) DNA replication, (C1) base/nucleotide excision repair, (D1) nuclear pore, (E1) nucleasome and (F1) xenobiotics metabolism over TC without WP treatment. Proteins with positive or negative FCs are up or downregulated, respectively, in later days. The shade defines |FC| < 1.05 or Benjamini-Hochberg (BH) p-value < 0.05. (A2-F2) Parallel pathway analysis for +/−WP samples over D9 → D15 and D9 → D22 durations. Same proteins from the two different treatment durations are connected by solid lines. Proteins with positive or negative FCs are up- or down-regulated, respectively, under +WP conditions. The areas of dots are proportional to the number of significant peptide sequences that can be assigned to a protein. To facilitate the visualization of proteins with small number of significant sequences, the scale is limited from 1 to 50 with no distinction of greater values. The shade defines |FC| < 1.05 or BH p-value < 0.05. (G) Viability of +/−WP samples over D9 → D22 period. Values were normalized to D9 RFU. N = 12 – 36. * p < 0.05, ** p < 0.005, **** p < 0.00005. (H) GST activity of +/−WP samples over D9 → D15 and D9 → D22 periods. N = 12 – 18. Absorbance, 340 nm; ΔA, 0–6min.
Figure 7.
Figure 7.
Abundance changes of Caco-2 protein in KEGG pathways depicting various apical, basolateral or paracellular processes. Cells with (+) or without (–) WP treatments were compared over D9 → D15 and D9 → D22 periods. Same proteins from the two different treatment durations are connected by solid lines. Proteins with positive or negative FCs are up- or down-regulated, respectively, under +WP conditions. (A) ABC transporters. (B) Proteasome. (C) Lysosome. (D) ECM-receptor interaction. (E) Fatty acid degradation. (F) Fatty acid metabolism. (G) Peroxisome. (H) PPAR. (I) Glycolysis/gluconeogenesis. (J) Oxidative phosphorylation. The shade defines |FC| < 1.05 or BH p-value < 0.05. ABC, ATP-binding cassette; ECM, extracellular matrix.
Scheme 1.
Scheme 1.
Overview of the study. (A) Demonstration of the feasibility in sensing subtle changes in protein abundances using shotgun proteomics. (B) Application of the procedure to examine the temporal proteome of Caco-2 monolayer with and without the stimulation of human WP.

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