Human umbilical cord blood proteome survey because large volumes of UCB are required to obtain sufficient stem cells for therapeu- tic purposes (Buchheiser et al. 2009; Forraz and McGuckin 2011). Hence, reducing the required volume for diagnostic purposes to a minimum is highly desirable. Moreover, we compared the identified proteins with known and proposed biomarkers to provide a short list of potential biomarkers that form a basis for the exploration of molecular mechanisms of exposure-disease relationships. Materials and Methods Chemicals. All chemicals were obtained from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA) with the following exceptions: Sequencing-grade modified porcine trypsin was obtained from Promega (Madison, WI, USA), and Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). UCB serum samples. The UCB serum samples were acquired from the cord blood cohort collection of the Baltimore Tracking Health-Related Environmental Exposures (THREE) study. This study was approved by the Johns Hopkins Medicine Institutional Review Board (IRB approval 04-04-22-02) and received a waiver from the Health Insurance Portability and Accountability Act (2002). The study showed U.S.-representative exposure levels (mean and maximum concen- trations) of a) perfluorooctanoate (PFOA): 1.6 ng/mL, 7.1 ng/mL; b) perfluorooctane sulfonate (PFOS): 5 ng/mL, 34.8 ng/mL; c) organochlorine pesticides (e.g., trans-non- achlor): 94 pg/mL, 185.5 pg/mL; d) trans- permethrin: 36.3 pg/mL, 34.8 ng/mL; e) polychlorinated biphenyls (PCBs) (e.g., di-ortho): 17.4 ng/g lipid, 176.5 ng/g lipid; and f) heavy metals (e.g., lead: 0.66 μg/dL, 15.5 gμ/dL; copper: 38.6 gμ/dL, 265 gμ/dL). Details on this cohort are described elsewhere (Apelberg et al. 2007a, 2007b; Herbstman et al. 2007; Neta et al.2011; Wells et al. 2011). Of this cohort study, 12 samples (eight male and four female) (Table 1) were ran- domly chosen for proteomic profiling. Using the Witter cord cradle (Witter et al. 2001), hospital-trained personnel collected the UCB by direct venipuncture of the umbilical vein, which assured that no maternal blood was present in the samples. In addition, analysis of the X:Y chromosome ratios in male newborns as described by Guerrero-Preston et al. (2010) further confirmed the absence of maternal blood. Up to five 10-mL UCB samples were collected per newborn and immediately stored at 4°C. Within < 3 hr, the refrigerated blood specimens were centrifuged at 1,000 × g for 15 min to collect the serum. Serum samples were then fractionated and stored in 2-mL polypropylene cryovials at –80°C. For pro- teomic analysis, frozen sample splits were shipped on dry ice to the Biodesign Institute at Arizona State University; the individual samples were thawed for the first time just before sample processing. Preparation of reference pool samples and immunodepletion. Aliquots of 100 Lμ of each of the 12 individual UCB samples were pooled to obtain a composite sample with a protein concentration of 79 mg/mL protein as determined by Bradford assay. Of this pool, 240 μL were taken and human serum albu - min (HSA) depleted using a Vivapure anti- HSA kit (VivaScience, Hannover, Germany) according to the manufacturer’s description. We chose not to further deplete the samples because several of the other highly abundant blood proteins often routinely depleted are either U.S. Food and Drug Administration (FDA)–approved adult blood biomark- ers (Anderson 2010) or proposed biomarker candidates for diverse diseases or exposures (Colquhoun et al. 2009; Ehmann et al. 2007; Ward et al. 2006). The HSA-depleted pro- teome fraction was then concentrated and desalted by ultrafiltration using the Vivaspin 500 concentrators (MWCO 3kDa; Sartorius, Goettingen, Germany). The total volume of the resultant composite sample was 150 μL, with a protein concentration of 33 mg/mL. Protein digest and sample fractiona­ tion. Proteins were denatured and reduced in 10 mM ammonium bicarbonate and 0.05% sodium dodecyl sulfate with 10 mM dithiothreitol at room temperature for 1 hr. Alkylation of proteins was accomplished by incubation in 40 mM iodoacetamide for 1 hr at room temperature in the dark. Ten micro- liters sequencing-grade modified porcine trypsine (1 mg/mL stock solution; Promega, Madison, WI, USA) was added, and the mix- ture was incubated at 37°C overnight. Tryptic digests (100–500 μg ) were then fractionated with the Agilent 3100 OFFGEL Fractionator using the 3100 OFFGEL Low Res Kit, pH 3-10 (Agilent Technologies, Santa Clara, CA, USA). The isoelectric focusing (IEF) was performed without ampholytes and glycerol according to the manufacturer’s instructions for peptide focusing. In short, the peptides were separated in a linear gradient of up to 8,000 V. The potential was kept at 8,000 V until 56,000 Vh was reached. The 12 IEF fractions were then extracted with 0.1% trif- luoroacetic acid in 50% methanol, vacuum- concentrated, and dissolved in 2% acetonitrile and 0.1% trifluoroacetic acid. Reverse­phase liquid chromatography (RP­LC ) separation and MS­analysis. For the RP nano-LC separation, a Tempo LC MALDI Spotting system (Applied Biosystems/ MDS SCIEX, Foster City, CA, USA) was used with a 2-μL injector loop and a Chromolith CapRod column (150 × 0.1 mm; Merck, Darmstadt, Germany). Separation was obtained by running a gradient at a Environmental Health Perspectives • volume 120 | number 5 | May 2012 2-Lμ/min flow rate. Solution A contained 2% LC-grade acetonitrile and 0.1% trifluoroacetic acid; solution B contained 98% acetonitrile and 0.1% trifluoroacetic acid. A 30-min gradient elution with the following param- eters was used: 2% B (0.5 min), 2%→40% B (0.5–15 min), 40%→65% B (15–22 min), 6 5 % → 8 0 % B ( 2 2 – 2 4 m i n ) , 8 0 % B (24–26 min), 80%→2% B (26–28 min), 2% B (28–30 min). The matrix-assisted laser desorption/ionization (MALDI) matrix solution (7 mg/mL recrystallized α-cyano- hydroxycinnamic acid, 0.1% trifluoro- acetic acid, 70% acetonitrile) was added postcolumn with a flow rate of 2 μL/min. Every 7 sec the combined eluate was auto- matically spotted onto a matrix prespotted stainless steel MALDI target plate (Applied Biosystems/MDS SCIEX). For calibration, 13 calibrant spots (ABI 4700 Mix) were added to each plate manually. All spotted samples were analyzed with a 4800 MALDI-tandem time-of-flight (TOF/TOF) mass spectrometer (Applied Biosystems/MDS SCIEX). First, MALDI-MS spectra were acquired over a mass range of m/z 800–4,000 in positive-ion reflec- tor mode using 70–500 laser shots/spectrum with a fixed relative laser power of 3,300 and a central biased spot search pattern. In each MS spectrum, up to 25 peaks were selected for MS/MS using an acquisition method that excluded ions with signal-to-noise (S/N) ratios of < 50. The precursor ion with the weak- est S/N ratio was acquired first to achieve the maximum signal intensity for low-abundance peptides. Tandem MS mode was operated using air as the collision-induced dissociation gas and enabled metastable ion suppressor Table 1. Statistics of selected UCB donors. No. of donors Characteristics Maternal race African American Caucasian Asian Maternal education ≤ High school diploma 1–4 years of college ≥ 5 years of college Health insurance status Private Medicaid Uninsured Body mass index Overweight Obese Substance abuse Smoking Reported diseases during pregnancy Anemia Asthma Thyroid condition Sexually transmitted disease Urinary tract infection Maternal age (years) Gestational age (days) 6 5 1 7 2 3 5 3 4 1 3 2 1 1 1 1 1 17–36 270–288 765