FNDC5 is produced in the stomach and associated to body composition

The fibronectin type III domain-containing protein 5 (FNDC5) discovered in 2002 has recently gained attention due to its potential role in protecting against obesity. In rat, no data exist regarding FNDC5 production and regulation in the stomach. The aim of the present work was to determine the expression of FNDC5 in the rat stomach and its potential regulation by body composition. The present data shows FNDC5 gene expression in the gastric mucosa. Immunohistochemical studies found FNDC5 immunopositivity in chief cells of gastric tissue. By the use of three different antibodies FNDC5 was found expressed in gastric mucosa and secreted by the stomach. The rate of gastric FNDC5 secretion parallels the circulating levels of FNDC5. The body fat mass increase after intervention with high fat diet coincided with a decrease in the secretion of FNDC5 from the stomach and a diminution in the FNDC5 circulating levels. In summary, the present data shows, for the first time, the expression of FNDC5 in the stomach of rats and its regulation by body composition, suggesting a potential role of gastric FNDC5 in energy homeostasis.


Methods and Results of Mass Spectrometry Assay.
FNDC5 immunoprecipitation : 1 mg of total protein was incubated with 2 µg of FNDC5 monoclonal antibody (catalog ab174833, Abcam, Cambridge, UK) overnight at 4ºC, followed by addition of 20 µL of 50% protein A/G-agarose beads (Santa Cruz Biotechnology) for 2 h at 4ºC. After incubation, beads were washed three times with RIPA buffer and three times with H 2 Omq. The pellet was loaded in a 10% SDS-PAGE gel and the bands were visualized using coomassie blue staining. Two bands (25 and 15 kDa) were selected to perform the FNDC5 identification assays by two different proteomics approaches: LC-MALDI/TOF assay or using higher sensitive equipment, 1D-nano LC ESI-MSMS.

In -gel protein digestion and sample preparation
Bands of interest from 1D gels were excised manually, deposited in 96-well plates and processed automatically in a Proteineer DP (Bruker Daltonics, Bremen, Germany) or subjected to a manual digestion. The digestion protocol used was based on Schevchenko et al.
[1] with minor variations: gel plugs were washed firstly with 50 mM ammonium bicarbonate and secondly with ACN prior to reduction with 10 mM DTT in 25 mM ammonium bicarbonate solution, and alkylation was carried out with 55 mM IAA in 50 mM ammonium bicarbonate solution. Gel pieces were then rinsed firstly with 50 mM ammonium bicarbonate and secondly with ACN, and then were dried under a stream of nitrogen. Proteomics Grade Trypsin (Sigma Aldrich), at a final concentration of 16 ng/μl in 25% ACN/50 mM ammonium bicarbonate solution, was added and the digestion took place at 37°C for 4 h. The reaction was stopped by adding 50%ACN/0.5%TFA for peptide extraction. The tryptic eluted peptides were dried by speed-vacuum centrifugation and storaged at -20°C for further analysis.
MS spectra were acquired in reflector positive-ion mode with a Nd:YAG, 355 nm wavelength laser, averaging 1000 laser shots and using 1296.685 peaks of angiotensin I (Sigma) as internal calibration. All MS/MS spectra were performed by selecting the precursors with a relative resolution of 300 (FWHM) and metastable suppression.
Peptide and protein identification were performed using the Protein Pilot software vs 4.0.80.85 (ABSciex) with Paragon Algorithm. MS/MS data was searched against the UniProt/Swiss-Prot database of protein sequences (release 2015-2; Swiss-Prot, Geneva, Switzerland). Searches were restricted to Rattus Novergicus taxonomy allowing carbamidomethyl cysteine as a fixed modification and oxidized methionine as variable modification. Both the precursor mass tolerance and the MS/MS tolerance were set at 30 ppm and 0.35 Da, respectively, allowing 1 missed tryptic cleavage site. Only proteins with a threshold >95% confidence (>1.3 unused score) were considered as positive hits.\

Liquid chromatography and triple TOF 5600 mass spectrometer analysis
Digested peptides of each sample was subjected to 1D-nano LC ESI-MSMS analysis using a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA) coupled to high speed Triple TOF 5600 mass spectrometer (AB SCIEX , Foster City, CA) with a Nanospray III source. The analytical column used was a silica-based reversed phase column C18 ChromXP 75 µm × 15 cm, 3 µm particle size and 120 Å pore size (Eksigent Technologies, AB SCIEX, Foster City, CA). The trap column was a C18 ChromXP (Eksigent Technologies, AB SCIEX, Foster City, CA), 3 µm particle diameter, 120 Å pore size, switched on-line with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 µl/min. The nano-pump provided a flow-rate of 250 nl/min and was operated under gradient elution conditions, using 0.1% formic acid in water as mobile phase A, and 0.1% formic acid in acetonitrile as mobile phase B. Peptides were separated using a 100 minutes gradient ranging from 2% to 90% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid). Injection volume was 5 µl.
Data acquisition was performed with a TripleTOF 5600 System (AB SCIEX, Foster City, CA). To detect lower abundance proteins such as FNDC5 protein, data was acquired using pseudo-MRM approach -an adaptation of the selected reaction monitoring (SRM) scanning mode-that consists of targeted acquisition of product ion spectra associated to unique and specific peptides from these proteins.
All data was acquired using an ionspray voltage floating (ISVF) 2300 V, curtain gas MS and MS/MS data obtained for individual samples were processed using Analyst® TF 1.7 Software (AB SCIEX, USA). Raw data file conversion tools generated mgf files which were also searched against the Rodentia UniProtKB/SwissProt database using the Mascot Server v. 2.5.0 (Matrix Science, London, UK). Search parameters were set as follows: carbamidomethylation of cysteines as fixed and oxidized methionines as variable modification. Peptide mass tolerance was set to 25 ppm and 0.05 Da for fragment masses, also 2 missed cleavages were allowed.

Results
We were not able to identify FNDC5 protein in either proteomic approaches. In the first assay we tried to identify FNDC5 using the commercial FNDC5 peptide as sample; however the LC-MALDI technology was not able to identify this protein. Therefore, we hypothesize that MALDI ionization approach may not be sensitive for FNDC5 detection. In order to improve the identification assay we performed a high sensitive approach, the triple TOF; however, we were unable to identify FNDC5. We tried a simple search approach or a RMN approach aimed to search the specific peptides obtained in the Silico tryptic digestion. As show in the attachment data, again we did not find FNDC5 in any of these approaches. We believe that the majority of the tryptic peptides obtained by the trypsin digestion might not be efficiently ionizated for its identification.