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. Author manuscript; available in PMC: 2016 Jun 2.
Published in final edited form as: Cell Metab. 2015 Jun 2;21(6):898–904. doi: 10.1016/j.cmet.2015.05.004

Detection of FGF15 in Plasma by Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) and Targeted Mass Spectrometry

Takeshi Katafuchi 1,7, Daria Esterházy 1,5,7, Andrew Lemoff 2, Xunshan Ding 1,3,6, Varun Sondhi 1, Steven A Kliewer 1,3,*, Hamid Mirzaei 2,*, David J Mangelsdorf 1,4,*
PMCID: PMC4454892  NIHMSID: NIHMS689408  PMID: 26039452

SUMMARY

Fibroblast growth factor 15 (FGF15) has been proposed as a postprandial hormone that signals from intestine to liver to regulate bile acid and carbohydrate homeostasis. However, detecting FGF15 in blood using conventional techniques has proven difficult. Here, we describe a stable isotope standards and capture by anti-peptide antibodies (SISCAPA) assay that combines immuno-enrichment with selected reaction monitoring (SRM) mass spectrometry to overcome this issue. Using this assay, we show that FGF15 circulates in plasma in an FXR and circadian rhythm-dependent manner at concentrations that activate its receptor. Consistent with the proposed endocrine role for FGF15 in liver, mice lacking hepatocyte expression of the obligate FGF15 co-receptor, β-Klotho, have increased bile acid synthesis and reduced glycogen storage despite having supraphysiological plasma FGF15 concentrations. Collectively, these data demonstrate that FGF15 functions as a hormone and highlight the utility of SISCAPA-SRM as a sensitive assay for detecting low abundance proteins in plasma.

INTRODUCTION

FGF15 is a member of a subfamily of FGFs, including FGF19, FGF21 and FGF23, which function as hormones (Beenken and Mohammadi, 2009). FGF15 is the mouse ortholog of human FGF19, although they share only 50% amino acid identity (Katoh, 2003). Expression of both Fgf15 and FGF19 is stimulated in the ileum by the bile acid receptor, FXR, during the postprandial reuptake of bile acids (Inagaki et al., 2005; Lundasen et al., 2006). Current evidence suggests that both FGF15 and FGF19 are secreted from enterocytes and transported to liver, where they bind and activate a heteromeric receptor complex composed of fibroblast growth factor receptor 4 (FGFR4) and β-Klotho in hepatocytes. This results in transcriptional repression of CYP7A1, which encodes the rate-limiting enzyme in bile acid synthesis (reviewed in (de Aguiar Vallim et al., 2013; Potthoff et al., 2012)). Genetic loss and gain of function studies of various components in this signaling pathway, including FXR, FGF15 and FGFR4, have demonstrated the pathway’s physiologic significance in bile acid homeostasis in mouse (Inagaki et al., 2005; Yu et al., 2000). FGF19 is readily detectable in human serum by standard ELISA, and its circulating levels are inversely correlated with bile acid synthesis (Lundasen et al., 2006; Walters et al., 2009). However, for unknown reasons, FGF15 is a weak antigen, and ELISAs to detect FGF15 in the blood have not been available, leading to questions about whether it is a true hormone (Angelin et al., 2012).

Here we describe the application of a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA) (Anderson et al., 2004) that combines the power of targeted mass spectrometry (Mirzaei et al., 2013) and immunopurification to definitively measure FGF15 concentrations in plasma. By employing SISCAPA together with the hepatocyte specific knockout of β-Klotho, we provide unequivocal evidence for the role of intestine-derived FGF15 in the gut-liver endocrine axis that governs bile acid synthesis. Further, this work highlights the potential of SISCAPA for studying low abundance endocrine factors that are otherwise refractory to detection.

RESULTS AND DISCUSSION

The SISCAPA-SRM assay

Plasma has one of the most complex proteomes both in terms of the number of proteins and their range of concentrations. Because the dynamic range of protein concentrations in plasma can range over 10 orders of magnitude (Hortin and Sviridov, 2010), achieving deep coverage of the plasma proteome has been difficult. Selected reaction monitoring (SRM) is currently the most sensitive mass-spectrometry technique for the detection of peptides in complex mixtures (Picotti et al., 2013). This technique, which relies on quadrupole mass analyzers, achieves superior sensitivity by sequential ion and fragment ion filtering instead of trapping. However, even SRM is limited by the ionization efficiency of peptides in complex mixtures. For this reason, SISCAPA technology was developed to achieve even greater sensitivity by combining the power of immunopurification of peptide surrogates of target proteins and SRM (Anderson et al., 2004).

Western blot assays and ELISAs commonly used for protein detection and quantification rely on antibodies. In cases where high-affinity antibodies cannot be raised and where SRM is not adequately sensitive, SISCAPA is a remaining option. SISCAPA uses peptide-specific antibodies to enrich peptides from proteins of interest for SRM analysis (Figure 1A). Antibodies are developed against those peptides unique to the target protein (proteotypic peptides) and with the best ionization efficiency and chromatographic behavior. These antibodies are then used to enrich the target peptides from a plasma digest. Stable heavy isotope-labeled peptide(s) are added to the plasma digest prior to peptide immunopurification as internal standards for SRM quantification.

Figure 1. The SISCAPA-SRM assay.

Figure 1

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(A) Workflow used for the development of the FGF15 SISCAPA-SRM assay. His-tagged FGF15 was purified from the lysate of HEK293 cells overexpressing the target. The affinity-purified sample was then digested and analyzed by shotgun MS for identification of peptides generated from FGF15. The three peptides (all proteotypic) with highest ion intensity and best chromatographic behavior were then selected and chemically synthesized. SRM alone was performed for plasma spiked with heavy labeled versions of the tryptic peptide. Rabbit antibodies were raised against the selected peptides and used for peptide enrichment from plasma digests. Only one of the target peptides was chosen for SRM after the affinity enrichment due to differences in antibody affinities and peptide ionization efficiencies. A heavy-isotope coded version of the detected peptide was added to the plasma digest prior to enrichment as an internal standard (IS) for absolute quantification.

(B) SRM traces are shown for both the light (blue) and heavy (red) y ions of FGF15-176 peptide (SFFETGDQLR) antibody-enriched from the digest of the plasma collected from wild-type (WT) and Fgf15-KO mice. Heavy (H) and light (L) ions are labeled y5, y6, and y8.

Detection of FGF15 in plasma by SISCAPA-SRM

As a first step towards developing a SISCAPA-SRM assay for FGF15, we determined which FGF15 peptides to monitor by SRM. FGF15 was expressed in HEK293 cells and enriched by nickel-affinity chromatography (Inagaki et al., 2005). The eluate was then digested with trypsin and analyzed by shotgun mass spectrometry. Over 700 proteins were detected in this purified sample based on a search of the data with CPFP version 2.0.3. (Trudgian and Mirzaei, 2012; Trudgian et al., 2010), and are listed in Table S1. Three proteotypic FGF15 peptides, FGF15-52 (LQYLYSAGPPYVSNXFLR), FGF15-176 (SFFETGDQLR) and FGF15-188 (MFSLPLESDSMDPFR), were detected (19.3% sequence coverage).

The SRM method was developed by determining the top seven transitions for heavy-labeled FGF15-52, -176, and -188 peptides based on injection of a mixture containing 50 fmol of each peptide. Once selected, the corresponding “light” endogenous FGF15 peptide transitions were generated by making the necessary m/z adjustments to the transitions. The liquid chromatography (LC) method was optimized by injecting spiked plasma samples and using 15, 30, and 45 minute gradients for separation to determine what gradient length provided the optimal combination of chromatographic separation and sensitivity. A 45 minute gradient resulted in minimal peak broadening relative to the shorter gradients and provided better contaminant separation, and was therefore used for the subsequent SRM analyses. The limit of detection (LOD) for FGF15 in plasma based on this SRM analysis, assuming complete tryptic digestion of FGF15, was determined to be 42.3 ng/mL. Additionally, the LOD for the FGF15-176 peptide by SRM was 5 times lower than that for the FGF15-188 peptide and 36 times lower than that for FGF15-52 peptide. However, we were unable to directly detect the three FGF15 peptides by SRM in plasma from mice, even after treatment for 6 hours with the potent, selective FXR agonist, GW4064 (Goodwin et al., 2000). Thus, based on the LOD of SRM, the level of FGF15 in plasma was predicted to be below 42.3 ng/mL.

To overcome this limitation, we generated rabbit polyclonal antisera against each of the three FGF15 peptides for the purposes of enrichment. Antibodies against each peptide were purified by peptide affinity chromatography and evaluated by ELISA. Antibodies against FGF15-176 had the highest affinity for their cognate peptide (Figure S1), coinciding with the lowest LOD of this antigen. The FGF15-176 peptide was also the best performing peptide based on ionization efficiency and detectability by SRM alone. Thus, the FGF15-176 antiserum was used to develop the SISCAPA-SRM assay (Figure 1A).

For detection of FGF15 in plasma, 30 μl of plasma were digested overnight with trypsin. Twenty femtomoles of labeled peptide were added for quantification, and tryptic peptides were purified using reversed-phase sorbent cartridges. For antibody immobilization, FGF15 antiserum was mixed with 10 μl of Protein G agarose beads (approx. 50% slurry) and 25 μl of PBS, and incubated for 45 min at room temperature with gentle shaking. Affinity-purified FGF15-176 antibodies were not used because the preparations contained detectable levels of co-eluted peptide from the affinity matrix, which would have contributed background to the SISCAPA-SRM assay. Moreover, as demonstrated below, the unpurified antiserum provided adequate sensitivity. The antibody-Protein G beads were washed twice with 0.2 M triethanolamine/PBS (pH 8.5) and the antibodies cross-linked to the beads with dimethyl pimelimidate (DMP). The conjugated beads were mixed with the sample in PBS containing 0.04% rapigest and incubated at 4°C with gentle shaking overnight. The peptides were eluted the following day using 5% acetic acid. The eluate was subjected to solid-phase extraction on an Oasis HLB plate and eluted with 40% ACN, 0.1% TFA in order to extract the FGF15-176 peptide without elution of more hydrophobic species that contaminate the LC/MS system. The eluate was dried and reconstituted for SRM analysis. Although SISCAPA enriches FGF15 for LC/MS analysis, a 45 minute HPLC gradient was used to distinguish the FGF15-176 peptide from non-specifically bound peptides with similar retention times.

We first tested the FGF15 SISCAPA-SRM assay using left ventricular plasma from fed wild-type and Fgf15-knockout (KO) mice (Wright et al., 2004). Quantification by SISCAPA-SRM revealed the presence of FGF15 in plasma from WT mice but not Fgf15-KO mice (Figure 1B). The LOD for FGF15 using the SISCAPA-SRM assay was 0.1 ng/mL (S/N = 3). We then evaluated FGF15 mRNA and plasma protein levels in fasted wild-type and Fgf15-KO mice treated with either vehicle or the FXR agonist, GW4064, for 6 hours. As expected, GW4064 treatment increased Fgf15 mRNA in ileum of wild-type mice whereas no Fgf15 mRNA was detected in Fgf15-KO mice (Figure 2A). Notably, FGF15 protein increased from 0.8 ng/ml in plasma from vehicle-treated mice to 6.3 ng/ml in plasma from GW4064-treated mice (Figure 2B). As expected, FGF15 was not detected in plasma from either vehicle or GW4064-treated Fgf15-KO mice (Figure 2B). The GW4064-induced increases in ileal Fgf15 expression and plasma FGF15 protein levels were coincident with an FGF15-dependent decrease in hepatic expression of Cyp7a1 (Figure 2C). Fgf15 mRNA was not detected in liver under any of these conditions (data not shown). As expected, GW4064 administration increased expression of the FXR target genes Shp and Insig2 in liver of wild-type mice (Figure S2). Interestingly, these genes were not induced by GW4064 treatment of Fgf15-KO mice, suggesting that FGF15 contributes to the induction of at least some FXR target genes in liver. Indeed, FGF19 treatment increases SHP expression in rat hepatocytes (Bhatnagar et al., 2009). However, Shp overexpression is not sufficient to efficiently repress Cyp7a1 in mice (Kir et al., 2012), indicating that FGF15/19 must regulate additional pathways required for the repression of bile acid synthesis.

Figure 2. Quantification of plasma FGF15 upon FXR activation.

Figure 2

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(A–C) WT and Fgf15-KO mice were treated with vehicle (open bars) or 100 mg/kg GW4064 (filled bars) for 5h after a 5h fast.

(A) Ileal Fgf15 mRNA expression.

(B) Plasma FGF15 levels quantified by SISCAPA.

(C) Hepatic Cyp7a1 mRNA expression.

(D) ERK2 phosphorylation upon stimulation with recombinant FGF15. Serum-starved H4IIE rat hepatoma cells were stimulated for 10 min with the indicated concentration of recombinant mouse FGF15. The phosphorylated and total ERK2 (P-ERK2 and T-ERK2, respectively) levels were quantified using ImageQuant LAS4000 system.

Data are shown as the mean ± SEM. n = 5–6/group. *p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle. n.d., not detected.

To determine whether FGF15 activates its receptor complex at its circulating concentrations, we performed dose response analysis using rat H4IIE hepatoma cells, which express both FGFR4 and β-Klotho, and monitored ERK2 phosphorylation, which is a well-established marker of FGFR4/β-Klotho activation (Kurosu et al., 2007; Wu et al., 2007). Partially-purified recombinant FGF15 increased ERK2 phosphorylation with an EC50 of ~1 ng/ml (Figure 2D). Thus, FGF15 activates its receptor complex at concentrations consistent with those detected in plasma.

To determine the diurnal variation in FGF15 levels, ad libitum fed C57BL/6 male mice were analyzed every 3h over a 24h period. Serum FGF15 protein levels mirrored the expected diurnal expression of ileal Fgf15 mRNA, which peaked at 1300h and was lowest at 1700–1900h (Figure 3A, B). As predicted, the diurnal regulation of FGF15 was reciprocal to the level of hepatic Cyp7a1 expression (Figure 3C). Taken together, these data validate the SISCAPA-SRM assay; demonstrate unequivocally that FGF15 circulates in the blood at physiologically relevant concentrations; and show that FGF15 mRNA and protein levels correlate with decreased bile acid synthesis.

Figure 3. Coordinate diurnal regulation of FGF15 and CYP7A1.

Figure 3

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(A) Ileal Fgf15 mRNA levels at the indicated times.

(B) Plasma FGF15 protein levels at the indicated times.

(C) Hepatic Cyp7a1 mRNA levels at the indicated times.

Ad lib-fed C57BL/6 mice (n = 5/group, 7−9 weeks old) were acclimated for 1 week, and tissues were harvested every 3h at indicated time points. Lights were turned on at 0700 and off at 1900. Data are shown as the mean ± SEM. *p < 0.05, ***p < 0.001 compared to the lowest measured value.

FGF15 acts directly on the liver to suppress bile acid synthesis

To test whether circulating FGF15 acts directly on the liver, we crossed mice with a floxed allele of the β-Klotho gene (Klbfl/fl) with albumin-Cre mice to generate Klbtm1(alb) mice in which Klb expression was selectively eliminated in liver but not other tissues, including brown and white adipose tissue depots (Figure 4A). Groups of control Klbfl/fl and Klbtm1(alb) mice were then administered either vehicle or FGF15. There were several important differences between Klbfl/fl and Klbtm1(alb) mice. First, basal Cyp7a1 expression was increased 4-fold in liver of Klbtm1(alb) mice (Figure 4B), and there was a corresponding increase in bile acid pool size (Figure 4C). There was also a trend towards increased Cyp8b1 expression in the Klbtm1(alb) mice (Figure 4B), which likely accounts at least in part for the increased fraction of cholic acid in the bile acid pool. Second, the increase in hepatic Cyp7a1 expression in Klbtm1(alb) mice occurred despite marked increases in Fgf15 mRNA levels in ileum (Figure 4D) and circulating FGF15 levels (Figure 4E). Third, whereas administration of exogenous FGF15 reduced Cyp7a1 mRNA levels in control Klbfl/fl mice, it had no effect in Klbtm1(alb) mice (Figure 4B). Finally, Klbtm1(alb) mice had reduced hepatic glycogen concentrations (Figure 4F), consistent with the established role of FGF15 in stimulating glycogen synthesis in liver (Kir et al., 2011). Together, these data demonstrate that FGF15 acts as a hormone on liver to suppress bile acid synthesis and to stimulate glycogen synthesis, and that these effects require hepatocyte specific expression of β-Klotho.

Figure 4. Hepatic expression of β-Klotho is required for normal bile acid and carbohydrate metabolism.

Figure 4

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(A) Klb gene expression in tissues from ad libitum fed WT (open bars) and Klbtm1(alb) (filled bars) mice (n.d., not detected).

(B) Hepatic Cyp7a1 and Cyp8b1 mRNA levels in ad libitum fed WT and Klbtm1(alb) mice treated with vehicle (open bars) or 1 mg/kg FGF15 (filled bars). Mice were injected with FGF15 when the lights were turned on and were killed 6h later.

(C) Bile acid pool size in WT and Klbtm1(alb) mice as in (B) (***p < 0.001 comparing total pool sizes).

(D) Fgf15 mRNA levels in ileum from same mice as in (B).

(E) Plasma FGF15 levels from same mice as in (B).

(F) Hepatic glycogen levels in same mice as in (B).

Data are shown as the mean ± SEM; n = 4–6. *p < 0.05, **p < 0.01, ***p < 0.001.

In summary, using SISCAPA-SRM, we demonstrate unequivocally that FGF15 acts as an FXR-regulated hormone to modulate bile acid synthesis and glycogen storage in liver. This study highlights the sensitivity and utility of the SISCAPA-SRM approach, which is likely to be broadly applicable for measuring other low abundance proteins. Overall, there was a strong correlation between FGF15 mRNA levels in ileum and plasma FGF15 concentrations. The circadian pattern of regulation of FGF15 observed in mouse was commensurate with that observed for FGF19 in humans (Lundasen et al., 2006). Moreover, the concentration of FGF15 measured in plasma is consistent with that required to activate its receptor. Finally, the striking induction of FGF15 mRNA and circulating protein concentrations in the Klbtm1(alb) mice demonstrates that this enterohepatic hormonal signaling pathway is essential for normal feedback regulation of bile acid homeostasis.

EXPERIMENTAL PROCEDURES

FGF15 peptide synthesis and antisera

Peptide synthesis and rabbit antisera generation against FGF15 fragments FGF15-52 (LQYLYSAGPPYVSNXFLR), FGF15-176 (SFFETGDQLR) and FGF15-188 (MFSLPLESDSMDPFR) were performed by Bethyl Laboratories Inc. The peptides were conjugated to KLH, and Complete Freund’s Adjuvant was used to elicit an immune response. Antibodies against the synthetic peptide antigens were purified by peptide affinity chromatography by Bethyl Laboratories Inc. and evaluated by measurement of binding activity to the serially diluted peptide plated onto an ELISA plate. Peptides were serially diluted with 100 mM Na2CO3 (pH 9.6) and applied in a 100 μl volume to 96-well Nunc high-binding plates (Thermo Fisher Scientific, Waltham, MA) at 4°C for 12h. Total amounts of peptides applied to the plates ranged from 0.001 nmol to 1000 nmol/well (Figure S1). After washing 3 times in PBS containing 0.1% Tween 20 (PBS-T), plates were blocked with PBS-T containing 1% BSA (300 μL/well) at room temperature for 3h. Antibodies (1 mg/ml) against the corresponding peptides were diluted 1:1500, 1:15000, and 1:150000 with PBS-T containing 1% BSA, added to plates (100 μL/well) and incubated overnight at 4°C. Plates were washed 3 times with PBS-T and 100 μL of anti-rabbit IgG conjugated with horseradish peroxidase (1:1,000 dilution, Millipore) were added to the wells for 2h at room temperature. Plates were washed 5 times with PBS-T, and 50 μL/well of 3,3′,5,5′-tetramethylbenzidine solution (Thermo) were added followed by 0.18 M H2SO4 (50 μL/well). Antibody binding to peptides was quantified by measuring absorbance at OD450. Stable heavy-isotope labeled peptides were synthesized by 21st Century Biochemicals Inc. (Marlboro, MA) with purities of >85% as determined by HPLC. All peptides were synthesized with a C-terminal [13C6,15N4] arginine, and all cysteines were carbamidomethylated. These peptides were used without further purification.

Immunopurification of FGF15 peptides

Thirty μL of mouse systemic plasma were mixed with the same volume of 100 mM Tris (pH 8.0) and 60 μL of trifluoroethanol, and mixed vigorously for 30 seconds. Following the addition of Tris (2-carboxyethyl) phosphine HCl (5 mM final concentration) and reduction of the samples at room temperature for 30 min with gentle agitation, iodoacetamide (2.5 mM final concentration) was added and the samples were alkylated at room temperature for 30 min in the dark. Trypsin digestion was performed by adding 1.08 mL of 100 mM Tris-HCl (pH 8.0) and 40 μL of a 1 mg/mL trypsin solution (proteomics grade trypsin, Sigma) and incubating the samples at 37°C overnight. The digestion was completed by adding the same amount of the trypsin solution and incubating the samples for 4h at 37°C, and the reaction was terminated by addition of 13 μL each of formic acid (~50%) and 10% trifluoroacetic acid (TFA). Complete digestion was confirmed by SDS-PAGE/silver stain analysis. Twenty fmol of stable isotope-labeled peptide were added prior to purification, for subsequent quantification and to account for variation in sample recovery. The trypsin-digested peptides were eluted from an Oasis HLB plate with 80% acetonitrile containing 0.1% TFA, and the solvent was evaporated in a SpeedVac concentrator. The samples were solubilized in 50 μl PBS containing 0.04% rapigest and sonicated.

For antibody immobilization reactions, 15 μl of FGF15 peptide fragment antiserum were mixed with 10 μl of Protein G agarose beads (50% slurry; Santa Cruz) and 25 μl of PBS, and incubated for 45 min at room temperature with gentle shaking. The antibody-Protein G beads were washed twice with 0.2 M triethanolamine/PBS (pH 8.5) and conjugated with 25 mM dimethyl pimelimidate (DMP) at room temperature for 30 min with gentle shaking. The excess DMP was quenched by washing the conjugated beads twice with 0.2 M ethanolamine/PBS (pH 8.0) for 5 min. The conjugated beads were rinsed three times with PBS containing 0.04% rapigest, and 50 μl of antibody beads (20% slurry) were mixed with the sample reconstituted as described above and incubated at 4°C with gentle shaking overnight. The beads were rinsed twice with PBS containing 0.04% rapigest, three times with PBS and once with 0.1X PBS. The peptides were eluted from the beads with 30 μl of 5% acetic acid. After 5 minutes, the samples were centrifuged and the supernatant containing the enriched peptides was dried in a SpeedVac concentrator and reconstituted in 100 μL of 0.5% TFA. This sample was incubated at 37°C with shaking for 45 min and then centrifuged at 13,000 rpm for 10 min to remove insoluble materials. The supernatant was cleaned by solid phase extraction on a Waters Oasis HLB plate, eluted with 40% ACN, 0.1% TFA, and dried in a SpeedVac concentrator. The sample was reconstituted in 6 μL of 98% water, 2% acetonitrile, 0.1% TFA for mass spectrometry analysis.

MS analysis

The FGF15 enriched samples were analyzed using a Q Exactive mass spectrometer (Thermo Scientific, Bremen) coupled to an Ultimate 3000 RSLCnano HPLC system (Dionex, Sunnyvale CA). Peptides were loaded onto a 75 μm i.d. × 50 cm column packed in-house with a reverse-phase material ReproSil-Pur C18-AQ, 1.9 mm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Peptides were eluted with a gradient comprised of 1–41% B in 40 minutes and 41–80% B in 10 minutes, where mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The column temperature was maintained at 60°C using a butterfly heater (Phoenix S&T, Inc. Chester, PA). The shotgun analysis was performed using a data-dependent top 20 method, with the full-MS scans acquired at 70 K resolution (at m/z 200) and MS/MS scans acquired at 17.5 K resolution (at m/z 200). The under-fill ratio was set at 0.3%, with a 3 m/z isolation window and fixed first mass of m/z 100 for the MS/MS acquisitions. Charge exclusion was applied to exclude unassigned and charge +1 species, and dynamic exclusion was used with duration of 7 s. The data were then searched using CPFP version 2.0.3. (Trudgian and Mirzaei, 2012; Trudgian et al., 2010)

SRM

The top seven transitions for each heavy-labeled peptide were determined by monitoring peak areas for all singly charged b and y ions below m/z = 1250 and for all doubly and triply charged peptide ions below m/z = 1000, for a 1 μL injection of 50 fmol of a mix of each of the three heavy-labeled peptide standards. These data were analyzed using Skyline v1.4 (http://skyline.maccosslab.org) (MacLean et al., 2010), and collision energies and declustering potentials were generated by the software without additional user optimization. Lists of the transitions, retention times, declustering potentials, and collision energies used are given in Table S2. Transitions that had interference from impurities or noise peaks were not included when performing peptide quantifications.

Spiked plasma samples were separated on a Dionex Acclaim PepMap100 reverse-phase C18 column (75 mm × 15 cm) using an Ultimate 3000 RSLCnano HPLC system. The HPLC was controlled using Chromeleon Xpress (version 6.8 SR10) and Dionex Chromatography MS Link v. 2.12. Separation of peptides was carried out at 200 nL/min using a gradient from 0–25% B for 45 min, 25–35% B for 10 min, and 35–80% B for 5 min, where mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Mass spectrometric analysis was performed on an AB Sciex (Foster City, CA, USA) 6500 QTRAP mass spectrometer in positive-ion low-mass mode, using a NanoSpray III source with a New Objective precut 360m PicoTip emitter (FS360-20-10-N20-10.5CT). The source settings were the following: Curtain gas = 35, ion spray voltage = 2550, ion source gas 1 = 6. Analyst Software v.1.6. was used to run the mass spectrometer. SRM data were analyzed using Multiquant v.2.1 (AB Sciex).

To determine the LOD of FGF15 by SRM alone (without antibody enrichment), we prepared a sample containing 100 μg of mouse plasma digest spiked with 100 fmol of heavy-labeled FGF15-176. Mouse plasma digest was used to account for the matrix effect on the LOD for FGF15 measured by SRM. We injected 2 μg of spiked plasma digest onto the column and based on the signal we extrapolated the LOD (S/N=3) for FGF15 to be 75 amol, which translates to 42.3 ng/mL in plasma, assuming that FGF15 was completely digested and the measurements were within the linear dynamic range of the instrument. The LOD for SISCAPA is determined by many factors, including the LOD of the SRM method, the anti-peptide antibody affinity for the targeted peptide, the concentration of FGF15 in the sample and the sample size. We were able to routinely detect and quantify FGF15 by SISCAPA using 30 μl of mouse plasma, with an LOD of 0.1 ng/mL (S/N = 3), which is over 400 times less than the LOD for SRM analysis alone.

Mouse experiments

All experiments were approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center. Littermate male mice on a C57BL/6 background maintained on a 12 h light cycle (lights on at ZT0700) were used for all experiments. Fgf15-KO mice were generated previously (Wright et al., 2004) and maintained in our facility. The Klb gene was deleted from hepatocytes by crossing Klbfl/fl mice (Ding et al., 2012) with albumin-Cre mice (The Jackson Laboratory). Liver-specific disruption of Klb was confirmed by real time PCR. For FXR agonist studies, WT or Fgf15-KO mice were fasted 5 h before orally gavage with vehicle or GW4064 (100 mg/ml in 1% methylcellulose and 0.1% Tween-80). For recombinant FGF15 studies, 1 mg/kg of FGF15 was intraperitoneally injected into ad libitum fed WT or Klbtm1(alb) mice in a volume of 1 ml/100g body weight saline (see below for preparation and quantification of purified protein). Five hours later the mice were sacrificed by carbon dioxide inhalation and tissues were harvested. Plasma was harvested from the left cardiac ventricle using an insulin syringe with a 29-gauge needle (BD). The blood was transferred to an EDTA tube (Sarstedt) and centrifuged at 1,000 × g for 5 min. The plasma was transferred to a 1.5 ml microfuge tube (Eppendorf) and immediately frozen in liquid nitrogen. Tissues were directly harvested into Trizol reagent, disrupted and frozen in liquid nitrogen.

Bile acid measurement

Bile acid pool size was measured as described previously (Schmidt et al., 2010). Briefly, liver, gallbladder, intestines and attached mesentery were collected and bile acids extracted by homogenization and boiling. Individual bile acid compositions were determined by MS equipped with high-performance liquid chromatography using the following bile salts as standards: tauro-β-muricholate, tauroursodeoxycholate, taurohyodeoxycholate, taurocholate (TCA), glycocholate, taurochenodeoxycholate, taurodeoxycholate, glycochenodeoxycholate, glycodeoxycholate, and taurolithocholate. Tauro-β-muricholate was purchased from Steraloids Inc. All the other bile acids were purchased from Sigma.

Liver glycogen content

Hepatic glycogen concentrations were measured as described (Kir et al., 2011).

Recombinant FGF15

Full-length mouse FGF15 cDNA harboring 6 histidine residues at the C-terminus (6× His-tagged) was subcloned into the pLVX-IRES-ZsGreen lentiviral vector (pLVX-FGF15). The lentiviral particles were generated by transfecting pLVX-FGF15 together with Δ8.9 and VSVG plasmids into Lenti-X 293T cells (Clontech). Lenti-X 293T cells were infected with the virus, and 6× His-tagged FGF15 protein was purified using Ni-NTA resin (Qiagen). To accurately determine the concentration of FGF15 in this partially purified preparation, the recombinant protein was quantified by SISCAPA.

Hepatoma cell line experiments

H4IIE cells (ATCC) were maintained in Eagle’s MEM supplemented with 10% heat-inactivated FBS. Cells were seeded onto 24-well plates and maintained until they reached >70% confluence, at which point the culture medium was replaced with DMEM containing 20 mM Hepes (pH 7.3), and the cells were cultured overnight. Two hours prior to FGF15 treatment, the medium was replaced with fresh DMEM containing 20 mM Hepes (pH 7.3). Cells were treated with FGF15 for 10 min, the medium was removed, and 1× SDS-PAGE sample buffer containing 2% 2-mercaptoethanol was added. Total ERK2 and phosphorylated ERK2 in the cell suspension were quantified by western blot analysis using antibodies purchased from Cell Signaling.

RT-QPCR

Liver and ileum RNAs were extracted using Trizol (Invitrogen) according to manufacturer’s protocol. Adipose tissue RNA was purified using RNeasy lipid tissue RNA purification kit. Purified RNA was treated with DNase and reverse transcribed with Multiscribe (Invitrogen) in the presence of random hexamer. The following primers were used for QPCR of individual mouse gene expression. Cyp7a1: 5′-agcaactaaacaacctgccagtacta-3′, 5′-gtccggatattcaaggatgca-3′; Cyp8b1: 5′-gccttcaagtatgatcggttcct-3′, 5′-gatcttcttgcccgacttgtaga-3′; Fgf15: 5′-acgggctgattcgctactc-3′, 5′-tgtagcctaaacagtccatttcct-3′; Klb: 5′-gatgaagaatttcctaaaccaggtt-3′, 5′-aaccaaacacgcggatttc-3′. Relative mRNA levels were calculated using the comparative Ct method normalized to U36b4.

Statistical analysis

Statistical analyses were performed by 2-way ANOVA with post-hoc correction (GraphPad Prism) unless indicated otherwise. Data are presented as the mean ± SEM; p < 0.05 was considered significant.

Supplementary Material

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Acknowledgments

We thank Yuan Zhang, and Heather Lawrence for technical assistance. This work was supported by National Institutes of Health grant R01DK067158 (S.A.K. and D.J.M.), the Robert A. Welch Foundation (grant I-1558 to S.A.K. and grant I-1275 to D.J.M.), the Cancer Prevention and Research Institute of Texas (grants R1121 and RP120613 to H.M.), a Swiss National Foundation Early Postdoc Mobility Fellowship (D.E.), and the Howard Hughes Medical Institute (D.J.M.). X.D. is an employee and stockholder of NGM Biopharmaceuticals;

Footnotes

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SUPPLEMENTAL INFORMATION

Supplemental Information includes one figure and two tables.

AUTHOR CONTRIBUTIONS

T.K. conducted and analyzed all experiments involving mice, cell lines and preparation of samples for SISCAPA-SRM. D.E. initiated the project and designed, conducted, and analyzed experiments to develop FGF15 peptides and antisera and mouse experiments. X.D. generated Klbtm1(alb) mice. A.L. conducted and analyzed all MS experiments. V.S. performed the measurement and analysis of bile acids. H.M., S.A.K. and D.J.M. designed and supervised the project and wrote the paper.

The other authors have no conflicts of interest to declare.

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Associated Data

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Supplementary Materials

1
NIHMS689408-supplement-1.doc (22KB, doc)
2
NIHMS689408-supplement-2.xlsx (80.3KB, xlsx)
3
NIHMS689408-supplement-3.xlsx (10.3KB, xlsx)
4
NIHMS689408-supplement-4.pdf (87.5KB, pdf)

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