EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 43, No. 7, 427-435, July 2011 Discovery of the serum biomarker proteins in severe preeclampsia by proteomic analysis Jisook Park1*, Dong Hyun Cha2*, Soo Jae Lee1, further validate the candidate proteins with a quantita- Young Nam Kim3, Young Hwan Kim4 tive mass spectrometric method, selective reaction and Kwang Pyo Kim1,5 monitoring (SRM) and enzyme linked immune assay (ELISA) of serum samples collected from pregnant 1Department of Molecular Biotechnology women with severe PE (n = 8) or normal pregnant wom- Institute of Biomedical Science and Technology en (n = 5) was conducted. α2- HS-glycoprotein (AHSG), Konkuk University retinol binding protein 4 (RBP4) and α-1-micro- Seoul 143-701, Korea globulin/bikunin (AMBP) and Insulin like growth factor 2Department of Obstetrics and Gynecology binding protein, acid labile subunit (IGFBP-ALS) were Kangnam CHA Hospital confirmed to be differentially expressed in PE using CHA University SRM (P ＜ 0.05). Among these proteins, AHSG was Seoul 135-080, Korea verified by ELISA and showed a statistically significant 3Department of Obstetrics and Gynecology increase in PE samples when compared to controls. Inje University Pusan Paik Hospital Keywords: α2HS-glycoprotein; biomarkers; pre-eclamp- Busan 614-735, Korea sia; spectrometry, mass, electrospray ionization; pro- 4Division of Mass Spectrometry Research teomics; serum Korea Basic Science Institute Ochang 363-883, Korea 5Corresponding author: Tel, 82-2-458-7682; Introduction Fax, 82-2-452-5558; E-mail, [email protected] *These authors contributed equally to this work. Preeclampsia (PE) is a disorder defined by DOI 10.3858/emm.2011.43.7.047 new-onset hypertension and proteinuria after 20 weeks of gestation that can present as late as 4-6 Accepted 31 May 2011 weeks postpartum. PE affects 3-8% of all pregnancies Available Online 3 June 2011 and is a major cause of maternal morbidity and mortality (Noris et al., 2005). However, the under- Abbreviations: AHSG, α2-HS-glycoprotein; AMBP, α1-microglobulin/ lying pathogenesis of preeclampsia remains un- bikunin; CID, collision-induced dissociation; ESI, electrospary known, and the development of adequate bio- ionization; FDR, false discovery rate; IGFBP-ALS, insulin like growth markers for its diagnosis and prognosis have not factor binding protein, acid labile subunit; MS, mass spectrometry; yet been developed (Jauniaux et al., 2006). PE is PE, preeclampsia; RBP4, retinol binding protein 4; SRM, selective generally characterized as follows: 1) during the reaction monitoring early period of pregnancy, the placenta releases several growth factors into the maternal circulation, producing excessive systemic inflammation in Abstract response to ischemic hypoxia and oxidative stress (Borzychowski et al., 2006). 2), the generalized Preeclapsia (PE) is a severe disorder that occurs dur- endothelial dysfunction leads to maternal charac- ing pregnancy, leading to maternal and fetal morbidity teristic symptoms of preeclampsia such as hyper- and mortality. PE affects about 3-8% of all pregnancies. tension and proteinuria during the late period of In this study, we conducted liquid chromatography- pregnancy (Taylor, 1997). Although PE has been mass spectrometry/mass spectrometry (LC-MS/MS) studied for a long time, there are currently no reliable biomarkers to predict PE during early pre- to analyze serum samples depleted of the six most gnancy. abundant proteins from normal and PE-affected preg- Recently, many groups have been giving special nancies to profile serum proteins. A total of 237 pro- attention to serum proteins related to obesity, low teins were confidently identified with ＜ 1% false dis- grade inflammation and hypertriacylglycerolemia, covery rate from the two groups of duplicate analysis. which are common symptoms in PE (Smets et al., The expression levels of those identified proteins were 2006). Various serum proteins are considered to compared semiquantitatively by spectral counting. To 428 Exp. Mol. Med. Vol. 43(7), 427-435, 2011 contribute to the symptoms of PE, such as placental Table 1. Patient’s clinical characteristics growth factor (PlGF), vascular endothelial growth factor (VEGF), soluble vascular endothelial growth Clinical Pre-eclampsia Normal parameter (n = 8) outcome (n = 5) factor receptor-1 (sVEGFR-1), soluble fms-like tyrosine kinase-1(sFlt-1), placental protein 13 (PP- Maternal age (yrs) 30.75 32.4 13), pregnancy-associated plasma protein A (PAPP-A), (25-33) (28-38) human chorionic gonadotrophin (HCG), soluble Gestational age (wks ) 31.375 38.6 endoglin (sEng), alpha fetoprotein (AFP), insulin (27-39) (37-41) Birth weight (kg) 1.5025 3.184 like growth factor-1 and IGF-binding protein-1 (0.78-3.16) (2.71-3.91) (IGFBP-1) (Muller et al., 1996; Bersinger and Cesarean section (%) 8 5 Odegard, 2004; Audibert et al., 2005; Venkatesha (100%) (100%) et al., 2006; Baumann et al., 2007; Spencer et al., 2008; Teixeira et al., 2008). Various studies have shown that the expression levels of these proteins increase or decrease according to the disorder and its degree in PE (Baumann et al., 2007). However, it is not certain if these proteins would serve as new marker proteins for the diagnosis of PE in clinical practice. Because of these proteins relatively low specificities for the disease, further development is required for its application of clinical use. To enable commercial use, it is necessary over- Figure 1. Venn diagram of Identified proteins from both groups; (A) come challenges such as validation, reproducibility shows proteins identified with single peptide hit. Total of 111 proteins and specificity against novel marker proteins. shared between the PE and NC group. (B) represents the proteins that have multiple unique peptides. Seventy eight proteins were shared in Additionally, analysis of biological fluid that has both group. high complexity and a dynamic range requires ela- borate pre-preparation and more sensitive techni- ques (Hu et al., 2006). proteins for PE using data dependent LC-MS/MS For these reasons, in this study, we systematically for both identification and semi quantification. The compared the maternal serum proteome in women expression level of the differentially expressed with PE and normal pregnancy using LC-MS based proteins was further validated by the mass spec- proteomic technologies following depletion of highly trometric quantification method, SRM, and ELISA. abundant serum proteins, such as albumin, immu- We analyzed individual serum samples from five noglobulin, and transferring, etc. A total of 237 patients per group in duplicate by LC-MS/MS to serum proteins were confidently identified from identify the serum proteins (Table 1). In this anal- serum samples of women with PE and normal from ysis, the collected serum samples were treated 1 % false discovery rate searches (FDR). Among using a multiple affinity removal column (MARS) them, 97 proteins having multiple unique peptides containing six antibodies against albumin, IgG, IgA, were retained for group comparison. The differen- transferrin, haptoglobin, and alpha-1-antitrypsin (Ryu tial expression levels of the selected proteins were et al., 2010). After high abundant protein depletion, validated using a quantitative mass spectrometric the serum samples were digested using a sequence method, selective reaction monitoring (SRM) (Ji et grade trypsin for LC-MS/MS analysis to compare al., 2003) and enzyme linked immune assay (ELISA). the serum proteomes from women with PE and those from women undergoing normal pregnancy. The resulting MS/MS spectra were searched Results with SEQUEST algorithms using a composite target decoy database strategy to evaluate the confi- Mining of novel biomarkers from human biological dence of peptide (Ballif et al., 2005). We acquired fluids poses many challenges that must be over- 5984 and 5744 MS/MS spectra from the PE group come through a proteomics approach, such as the (n=5) and normal group (n=5), respectively. A total wide dynamic range in abundance and complexity of 237 proteins with 1 % FDR were assigned from of protein components. Accordingly, various attempts those spectra (Figure 1A). We limited the positive have been made to discover new biomarker pro- protein set to proteins identified by multiple pep- teins with clinical values (Whiteaker et al., 2007). tides. Among them, 97 proteins were matched with Herein, we endeavored to identify new marker 2 ≤ unique peptides (Figure 1B) (Table 2), and Biomarkers for preeclampsia serum 429 Table 2. Total identified proteins; A total of 237 proteins were these were subjected to further annotation and acquired from both groups with 1% FDR, with 97 of these validation. Consequently we identified 85 proteins proteins having multiple peptides. Each group had 85 and 90 and 90 proteins form serum samples of PE and serum proteins that were identified with multiple peptides serum samples of normal pregnancies, respec- tively. Among the 85 proteins identified in the PE Total number of Total number of groups, 49 proteins were identified in all five pa- IDed proteins IDed proteins Total number Group tients. Furthermore, 49 proteins were identified in (Multiple (Single of spectra peptide Hit) peptide Hit) all five patients among the 90 proteins identified in the normal group. PE 85 165 5984 Among the 97 confidently identified proteins, 78 Normal 90 185 5744 were identified in both groups. To screen potential Table 3. The fold changes in protein levels by spectral counts, which is one of the