Human milk whey proteins treated Caco-2 cells
Human milk whey proteins facilitate the development of enterocyte-like Caco-2 cells over time.
Sample Processing Protocol
At the indicated times, cells were harvested using a PBS-based enzyme-free cell dissociation buffer according to the manufacturer’s instructions, lysed by resuspension in RIPA buffer containing a protease inhibitor cocktail, followed by a freeze-thaw cycle. Protein concentration for the cell lysates was determined with BCA. The cell lysates were then prepared for tandem mass tag (TMT) labeling using the Pierce® TMT six-plex isobaric mass tagging kit (Thermo-Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions with slight modification. Briefly, cell lysates were dissolved in triethyl ammonium bicarbonate (TEAB), followed by reduction in tris(2-carboxyethyl)phosphine (TCEP) for 1 hour at 55 °C, and subsequent alkylation with iodoacetamide for 1 hour at room temperature. The alkylated proteins were then deglycosylated with PNGase F (1 U/50 µg protein) for 2 hours at 37oC, followed by acetone precipitation at -20 °C overnight. Following tryptic digestion overnight at 37oC, peptides were TMT labeled according to the manufacturer’s instructions. Each TMT 6-plex peptide mixture was pooled, lyophilized and resuspended in 200 L of 90% ACN/0.1% HOAc for injection onto a 4 mm i.d. × 10 mm WAX guard column (PolyWAX LP, 5 µm particle size, 1,000 Å pore size, PolyLC Inc., Columbia, MD) connected to a 2.1 mm i.d. × 200 mm WAX column (PolyWAX LP, 5 µm particle size, 300 Å pore size, PolyLC Inc.). Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) peptide separation was carried out via HPLC (U3000, Dionex, Sunnyvale, CA) at a flow rate of 200 µL/min. A gradient was started with 100% A (98% ACN, 0.1% HOAc) for 10 min and ramped to 28% B (30% ACN, 0.1% FA) over 68 min and held for 20 min, followed by a gradient ramped to 100% B over 20 min and then held at 100% B for 10 min. UV absorption was monitored at 280 nm. Thirty fractions with retention times ranging from 20 – 90 min were collected at 2 to 3-min intervals. Each fraction was dried under reduced pressure, reconstituted in 20 L of 0.1%FA, stored at -80 °C, and thawed at 4 °C when ready for nanocapillary liquid-chromatographic electrospray-ionization tandem mass-spectrometric (LC-ESI/MS/MS) analysis. LC-ESI/MS/MS analysis was conducted with a Q-Exactive mass spectrometer coupled to an EASY-nanoLC 1000 system (Thermo-Fisher Scientific). For each ERLIC fraction, 5 µL of sample was loaded onto a 75 m i.d. × 2 cm Acclaim PepMap 100 RP trap column (Thermo-Fisher Scientific). Peptide separations were carried out using an approximately 20-cm-long uncoated 75-m i.d., 15-m nanotip fused-silica column (New Objectives, Woburn, MA) packed in-house with 3-m C18 particles (Bruker-Michrom, Auburn, CA). The separation was started with 98% mobile phase A (0.1% FA) and to 40% B (ACN, 0.1% FA) in 150 min, followed by a 10-min wash at 60% B, with a flow rate of 300 nL/min. Full-scan mass spectra were acquired by the Orbitrap mass analyzer in the mass-to-charge ratio (m/z) of 300 to 1650 and with a mass resolving power set to 70,000. Ten data-dependent high-energy collision dissociations (HCD) were performed with a mass resolving power set to 17,500, a fixed first m/z 110, an isolation width of 4 m/z, and a normalized collision energy (NCE) setting of 30 with enabled stepped collision energy of 20% NCE. The maximum injection time was 100 ms for parent-ion analysis and 50 ms for product-ion analysis. Target ions already selected for MS/MS were dynamically excluded for 60 sec. An automatic gain control (AGC) target value of 3e6 ions was used for full MS scans and 1e5 ions for MS/MS scans. Duplicated LC/MS/MS analyses were carried out for each fraction. Only peptide ions with charge states of two or greater were selected for MS/MS interrogation.
Data Processing Protocol
MS/MS spectra with charges +2, +3 and +4 were analyzed using Mascot search engine (Matrix Science, London, UK; version 2.3.2). Mascot was set up to search against the human Uniprot/Swiss-Prot database (20,319 entries; version 2011_08) assuming the digestion enzyme was trypsin with a maximum of 1 missed cleavage allowed. The searches were performed with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine and deamidation of asparagine and glutamine were specified in Mascot as variable modifications. Iodoacetamide derivatization of cysteine and TMT 6-plex derivatizations of N-terminus and lysine were specified as fixed modifications. When searching against unlabeled peptides, TMT fixed modifications were disabled whereas acetylation of the N-terminus was specified as a variable modification. Peptide identities were accepted if they could be established at less than 1% probability of being a random match as specified by Mascot. A mass tolerance of 0.01 Da was chosen for peptide quantitation using TMT reporter ions. The protein ratios were calculated using the weighted average of the individual ratios of the peptides that can be assigned to that protein. Peptide identifications that can be assigned to more than one protein were removed from quantification. False-discovery rate (FDR) of peptides was estimated between 0.2 and 0.5% by searching against a decoy database. The identities of proteins with two or more significant decoy peptide matches were considered false. Protein FDR for each data set containing 30 ERLIC fractions was estimated between 0.1 and 0.3%.
Cundiff JK, McConnell EJ, Lohe KJ, Maria S, McMahon RJ, Zhang Q. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins. J Proteome Res. 2015 Nov 20 PubMed: 26586228