Itraq reagent

The protein concentrations were assessed a second time after trichloroacetic acid (TCA) precipitation overnight at 4°C using 1/3 volume of TCA, to ensure correct protein levels. For each sample 100 μg of protein was reduced using 5mM Tris-2-(carboxyethyl)-phosphine hydrochloride solution (TCEP; Pierce Biotechnology) and cysteine blocking was performed with 2mM methyl methanethiosulphonate (MMTS) solution (Sigma-Aldrich). Each sample containing 100 μg proteins, was digested using 10 μg trypsin (Promega) at 37°C overnight. Samples were then labelled using iTRAQ reagents (ABSciex) before being pooled into one mixture. The following iTRAQ labels were used for the transfected cells: iTRAQ labels 113 was used for the transfected cells with the pcDNA3.1+ empty plasmid; iTRAQ labels from 114 to 118 were used to label the differentially transfected cells with RXFP3 (114-0.5 μg_RXFP3-HA, 115-1 μg_RXFP3-HA, 116-2 μg_RXFP3-HA1, 117-5 μg_RXFP3-HA, 118-10 μg_RXFP3-HA). After labelling, the samples were pooled, dried down and dissolved with SCX buffer prior to SCX chromatography.

Isobaric tags for relative and absolute quantification (iTRAQ) was used to confirm the relative level of peptide expression in serum of ovarian tumor-bearing mice. All of the mouse serum samples (15 μl each) from each treatment group (injected with MSV-control siRNA or MSV-EphA2 siRNA) were pooled. To each serum pool, 10.2 μl of 5% TFA was added, and the peptides in solution were isolated by on-chip fractionation. The eluates from the group with the same treatment were combined, dried under vacuum centrifugation, and reconstituted in 0.5 M tetraethylammonium bromide (TEAB). Each of the iTRAQ reagents (AB-Sciex, Foster City, CA) was dissolved in 70 μl of ethanol for 2 min with vortexing and added directly to the dissolved peptides. The samples were mixed by vortexing and incubated for one hour at 25°C. The derivatized peptides were then pooled, dried under vacuum centrifugation, and re-dissolved in 0.1% TFA/water. Following desalting on a small C₁₈ column (2 mm × 1 cm), the peak fractions were left to air dry before being re-dissolved in buffer (1% formic acid in water containing 5 mM ammonium acetate). The pooled, derivatized and desalted peptides were subjected to LC-MS/MS analysis on a LTQ-Orbitrap Elite. The data were analyzed and quantified using Mascot (Matrix Science, London, UK, ver. 2.3) via Proteome Discoverer (Thermo Scientific, version 1.3).

About 100 µg proteins of each sample per tube were prepared. Then they were reduced with 12 mM (final concentration) dithiothreitol for 1 h at 37°C. Subsequently, protein alkylations were performed with 50 mM (final concentration) iodoacetamide for 1 h in dark at room temperature. Then the mixture was transferred to centrifugal units (VN01H02, Sartorius, Germany) and centrifuged at 12,000×g for 20 min, and then the filtrate was discarded. After centrifugation, 8 mM urea solution was added into the centrifugal units and centrifuged, this step was repeated twice. After that, 100 µL dilute buffer (50 mM triethylammonium bicarbonate) was added into the centrifugal units and centrifuged. Then 50 µL dilute buffer containing 2 µg modified trypsin (Promega) was added into the centrifugal units at 37°C overnight. After trypsin digestion, the resulting peptides were collected using centrifugation. Then the peptides were labeled with iTRAQ reagents (AB Sciex, USA) according to the manufacturer's instructions. For each sample of three time points (i.e., S1, S2, and S3) was iTRAQ labeled 3 times except S3. (i.e., 113-, 116-, 119- tags for S1, 3 replicates. 114-, 117-, 121- tags for S2, 3 replicates. 115-, 118- tags for S3, 2 replicates.)

Equal amounts of proteins from each group of different age cells (100 μg) were denatured with 2% sodium dodecyl sulfate (SDS) in 1 M tryethylammonium bicarbonate (TEAB) (ABSciex, Foster City, CA). The samples were then reduced for 1 h at 60 °C using 50 mM tris-(2-carboxyethy) phosphine (TCEP) (ABSciex), and cysteine-blocked with 84 mM iodoacetamide (Sigma-Aldrich) at room temperature in the dark for 30 min. The proteins were digested with spectrometry grade trypsin (Gold Mass, Promega, Madison,WI) at a concentration of 1:50 trypsin/protein for 16 h at 37 °C. Each peptide solution was labeled for 1.5 h at room temperature using the iTRAQ reagents previously reconstituted in 70 μL of ethanol, following the manufacture Protocol (ABSciex). The samples were labeled with iTRAQ reagents as follows: newborn: 119 and 121 as a control infant: 114; young: 116; pre-pubertal: 118; pubertal: 115; adult: 117. The reaction was stopped by adding deionized water, and the labeled samples were combined. The mixture was desalted using home-made stage-tips.

In order to further clarify the underlying mechanisms, we used iTRAQ to compare the changes of phosphorylated proteins. Pancreatic cancer cells treated with DMSO or gemcitabine for 48 h were harvested, protein extracted, quantified, reduced, cysteine blocked, digested and labeled, then the phosphorylated peptides were enriched and analyzed on mass spectrometer. Concretely speaking, proteins from the Patu8988 pancreatic cancer cells after gemcitabine treatment were precipitated with cold acetone for 2 h at −20 °C, pelleted by centrifugation at 12000 g, air-dried and dissolved into dissociation buffer. The total protein solution was quantified by BCA protein assay kit and protein lysate was added to the samples of high concentration to make the final concentration the same between the two groups. What’s more, the same amount and same volumes of proteins for two groups were given reductive alkylation and enzymatic hydrolysis. Then, the samples were labeled with isobaric tagging reagents according to protocols. iTRAQ reagents were obtained from AB SCIEX. The control group was labelled with 113 and 114; the gemcitabine treated group was labelled with 115 and 116. The results were obtained from three experiments. For every experiment, two independent replicate samples of control and gemcitabine treated group were included.