Collection of the material
Cyst fluids and blood were collected prospectively and consecutively from patients diagnosed from March 2001 to September 2010 with suspicious cystic pelvic tumors Patients were included when they were admitted for an operation to the section for gynecologic oncology surgery of Sahlgrenska University hospital, Gothenburg, Sweden. According to our protocol, blood samples were taken after anesthesia but before surgery, and cyst fluids were collected after removal of the cysts from the abdomen. All samples were put directly in 4°C for 15–30 minutes, centrifuged, aliquoted into eppendorf tubes, and stored in −80°C within 30–60 minutes after collection. Samples used in this study had experienced one freeze-thaw cycle. Removed tumors were examined by an experienced pathologist for histology and grade and staged (I-IV) according to FIGO standards. The local ethical committee at the University of Gothenburg approved the study, and each patient gave her informed, written consent.
To obtain a homogenous group of samples in the iTRAQ MS analysis, only serous ovarian adenomas and adenocarcinomas, the most common form of epithelial ovarian cancers, were included from our ovarian cyst fluid biobank. A total of 15 cyst fluid samples were analyzed with iTRAQ (Table
1). The following verification and validation included cyst fluid and plasma samples (n = 136) from 68 patients, with mix of all common ovarian histologies. We included 32 patients with benign cysts and 36 patients with EOC (Table
3). Seven samples from the iTRAQ analysis were included in the verification set, two benign and five malignant samples. In the validation step, the cohort consisted of tumors of different histologies, and serous carcinoma represented 50% of the malignant samples.
Sample preparation for MS analysis
Mass spectrometry analysis was performed at the Proteomic Core Facility at the University of Gothenburg. Our previous data showed that proteins, which are abundant in the blood, are even more abundant in cyst fluid
[18, 2]. Thus, beforehand removal of these proteins from the cyst fluid is required for the MS analysis to be able to detect potential tumor-specific biomarkers. In this study, we used a depletion method before MS and labeling by isobaric tag for relative and absolute quantitation (iTRAQ).
All 15 samples (50 μl each) were filtered using a 0.22 μm spin filter at 2000 rpm. The protein content was determined by Pierce BCA Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA). Depletion of human albumin and IgG were performed (25 μl of each sample) using the Qproteome Albumin/IgG Depletion Kit (Qiagen, Valencia, CA, USA). The protein concentration was determined once more by Pierce BCA Protein Assay (Thermo Fisher Scientific).
100 μg of each sample was withdrawn and diluted to 200 μl. Non-protein impurities were removed by quantitative precipitation clean-up using ProteoExtract® Protein Precipitation (Calbiochem, San Diego, CA, USA). The pellets were dissolved in iTRAQ® Dissolution Buffer with the addition of 1 μl 2% SDS (iTRAQ®, Applied Biosystems, Foster City, CA, USA), and the samples were digested with trypsin (Promega, Madison, WI, USA), reduced, and alkylated. All the 15 samples included in the analysis were pooled together and used as a standard for the iTRAQ analysis in each run. Each four-plex set consisted of one pooled standard sample and three different patient samples labeled with the iTRAQ® reagent 114, 115, 116, and 117 respectively, following the manufacturer’s instructions (Applied Biosystems).
Strong cation exchange chromatography (SCX) of iTRAQ-labeled peptides
The concentrated peptides were acidified by 10% formic acid and diluted with SCX solvent A (25 mM ammonium formate, pH 2.8, 20% acetonitrile [ACN]) and injected onto a PolySULFOETHYL A SCX column (2.1 mm i.d. × 10 cm length, 5 μm particle size, 300 Å pore size). SCX chromatography and fractionation was carried out on an ÄKTA purifier system (GE Healthcare, Buckinghamshire, UK) at 0.25 mL/min flow rate using the following gradient: 0% B (500 mM ammonium formate, pH 2.8, 20% ACN) for 5 min; 0-40% B for 20 min; 40-100% B for 10 min; and 100% B held for 10 min. UV absorbance at 254 and 280 nm was monitored while fractions were collected at 0.5 mL intervals and dried down in a SpeedVac. The peptide-containing fractions (10) were desalted on PepClean C18 spin columns according to the manufacturer’s instructions (Thermo Fisher Scientific).
LC-MS/MS analysis on LTQ-Orbitrap
The desalted and dried fractions were reconstituted into 0.1% formic acid and analyzed on a LTQ-Orbitrap XL (Thermo Fisher Scientific) interfaced with an in-house-constructed nano-LC system, described elsewhere
. Briefly, two-microliter sample injections were made with an HTC-PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) connected to an Agilent 1200 binary pump (Agilent Technologies, Palo Alto, CA, USA). The peptides were trapped on a precolumn (45 × 0.075 mm i.d.) and separated on a reversed phase column, 200 × 0.050 mm. Both columns are packed in-house with 3 μm Reprosil-Pur C18-AQ particles. The flow through from the analytical column was reduced by a split to approximately 100 nl/min, and the gradient was as follows: 0–5 min 0.1% formic acid; 6–103 min 7-32% ACN 0.1% formic acid; and 103–105 min 80% ACN 0.1% formic acid.
LTQ-Orbitrap settings were as follows: spray voltage 1.4 kV, 1 microscan for MS1 scans at 60 000 resolution (m/z 400), full MS mass range m/z 400–2000. The LTQ-Orbitrap was operated in a data-dependent mode, that is, one MS1 FTMS scan precursor ions followed by CID (collision induced dissociation) and HCD (high energy collision dissociation) MS2 scans of the three most abundant doubly or triply protonated ions in each FTMS scan. The settings for the MS2 were as follows: 1 microscan for HCD-MS2 at 7500 resolution (at m/z 400), mass range m/z 100–2000 with a collision energy of 50%; 1 microscan for CID-MS2 with a collision energy of 30%.
Database search and iTRAQ quantification
MS raw data files from all ten SCX fractions for one four-plex iTRAQ set were merged for relative quantification and identification using Proteome Discoverer version 1.1 (Thermo Fisher Scientific). A database search for each of the five sets was performed by Mascot search engine using the following criteria: homo sapiens in Swissprot version 57.15, MS peptide tolerance as 5 ppm, MS/MS tolerance as 0.05 Da, trypsin digestion allowing 2 missed cleavages with variable modifications; methionine oxidation, cysteine methylthiolation, tyrosine iTRAQ4plex (+144 Da) and fixed modifications; and N-terminal iTRAQ4plex, lysine iTRAQ4plex. The detected protein threshold in the software was set to 95% confidence, and identified proteins were grouped by those sharing the same sequences to minimize redundancy.
For iTRAQ quantification, the ratios of iTRAQ reporter ion intensities in MS/MS spectra (m/z 114.11-117.11) from the raw data sets were used to calculate fold changes (FC) between samples. Ratios were derived by Proteome Discoverer version 1.1 using the following criteria: fragment ion tolerance as 50 ppm for the most confident centroid peak; iTRAQ reagent purity corrections factors are used and missing values are replaced with minimum intensity. Only peptides unique to a given protein were considered for relative quantitation, excluding those common to other isoforms or proteins of the same family. The ratios were normalized to the mean value of the 50 ratios identified with highest number of peptides.
The protein concentrations of 68 cyst fluid and 68 plasma samples were determined with the Micro BCA protein assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific). The cyst fluid samples were diluted in H2O 1:10 and plasma samples 1:5, and 2.5 μl of each sample was diluted in (SDS) sample buffer with a reducing agent (Invitrogen). After heating at 70°C for 10 minutes, the samples were loaded on SDS-PAGE (NuPAGE 4–12% Bis-Tris Gel, Invitrogen Ltd., Paisley, UK) and separated by electrophoresis using MES SDS running buffer (Invitrogen). Proteins were transferred to polyvinyl difluoride membranes using the iBlot dry blotting system (Invitrogen). Membranes were blocked in 5% non-fat milk in 10 mM phosphate buffered saline (PBS) containing 0,05% Tween 20. The membranes were incubated overnight at 4°C with PBS containing 0,05% Tween 20 and the following primary antibodies: serum amyloid A-4 protein (SAA4) purified MaxPab mouse polyclonal antibody (1:1000, Abnova, Taiwan); astacin-like metalloendopeptidase (ASTL) (N-12) goat polyclonal (1:800, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); and 78 kDa glucose-regulated protein (GRP78) (N-20) goat polyclonal (1:500, Santa Cruz Biotechnology). Precision plus protein WesternC standards (Bio-Rad, Hercules, CA, USA) were used as molecular weight markers. Immunoreactivity protein was visualized by chemiluminescence using peroxidase-labeled secondary antimouse (1:10 000, GE Healthcare), secondary antigoat (1:15 000, Santa Cruz Biotechnology) detected with chemiluminescent ECL Advance (GE Healthcare). Immunoblotted membranes were exposed using a LAS-1000 (Fujifilm, Minato-ku Tokyo, Japan). Individual bands were quantified from the membrane images by densitometry using the Quantity One software program (Bio-Rad). An internal reference sample, the same on each blot, was used as a standard for quantification of bands detected in cyst fluid samples and was given the value 1
The normalized iTRAQ MS peak ratios were transformed to Log2 values. Protein entries with only a single peptide hit and proteins only detected in one or two sets, as well as various entries corresponding to IgG isoforms, were not included in the analysis. Differences between benign and malignant samples were compared using t-test and a list of significant results presenting proteins with p < 0.05 and at least a 1.8 fold change were generated.
For the validation assay, the statistical differences in protein expressions were calculated using the Mann–Whitney U test, and the relation between expressions measured with iTRAQ MS. Immunoblotting was evaluated with bivariate correlation using Spearman correlation coefficient. A value of p < 0.05 was considered to be significant.