Amniotic fluid(AF) samples were obtained by amniocentesis from women at 16–20 weeks of gestation undergoing prenatal diagnosis. Considering the interference of various factors, we screened eligible volunteers of similar ages (28 to 30 years with a median age of 29 years) in the 16th–20th weeks of pregnancy.

The 7 AF samples were precipitated overnight using 3 times the volume of ethanol at 4 °C. Then, after centrifugation at 10000×g for 30 min, the pellets were resuspended in lysis buffer (7M urea, 2M thiourea, 0.1M DTT, and 5mM Tris). The protein concentration of each sample was determined by spectrophotometry based on the Bradford method.The pooled AF samples were subjected to depletion of 14 high-abundance proteins using a 4.6×50mm Human 14 affinity LC column (Agilent, St Louis, MO) on a Waters HPLC system (Milford, MA). The separations were performed according to the manufacturer"s recommended procedures for the column and recommended loading capacity. The flow-through fraction and bound fraction were collected separately. The two fractions and the un-depleted sample (the original fraction) were subjected to the following sample handling and analysis.The 7 individual samples and pooled sample fraction (the flowthrough fraction, bound fraction and the original fraction) were digested using a filter-aided sample preparation (FASP) method .Protein samples (200 μg) were reduced with 20mM DTT at 95 °C for 5 min and then carboxyamidomethylated with 50mM IAM at room temperature in the dark for 45 min. Trypsin (4 μg) in 25mM NH4HCO3 was added to each protein sample for digestion overnight at 37 °C. After digestion, the resulting peptides were desalted on a Waters Oasis C18 solid-phase extraction column and lyophilized for HPLC separation.

The pooled sample fraction (the flow-through fraction, bound fraction and the original fraction) peptides were fractioned with a high-pH RPLC column from Waters (4.6 mm×250 mm, Xbridge C18, 3 μm). The samples were loaded onto the column in buffer A1 (H2O, pH=10). The eluted gradient was 5–30% buffer B1 (90% ACN, pH=10, flow rate=1 mL/min) for 60 min. The eluted peptides were collected at a rate of one fraction per minute. After lyophilization the 60 fractions were resuspended in 0.1% formic acid and concatenated into 20 fractions by combining fractions 1, 21, 41, etc.

Each fraction was analyzed using an RP C18 self-packing capillary LC column (75 μm×100mm, 3 μm). The eluted gradient was 5–30% buffer B2 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL/min) for 40min. A TripleTOF 5600 mass spectrometer was used to analyze the eluted peptides from LC. The MS data were acquired in high sensitivity mode using the following parameters: 30 data-dependent MS/MS scans per full scan; full scans were acquired at a resolution of 40,000 and MS/MS scans at 20,000; rolling collision energy, charge state screening (including precursors with +2–+4 charge state) and dynamic exclusion (exclusion duration 15 s); the MS/MS scan range was 100–1800m/z;and thescan time was 100 ms.

The MS/MS spectra were searched against the SwissProt human database (Homo sapiens, 20,267 sequences, 2013_07 version) from the SwissProt website (www.uniprot.org) using Mascot software version 2.3.02 (Matrix Science, UK).