Retina An iTRAQ-Based Quantitative Proteomic Analysis of Plasma Proteins in Preterm Newborns With Retinopathy of Prematurity Magdalena Zasada,1 Maciej Suski,2 Renata Bokiniec,3 Monika Szwarc-Duma,3 Maria Katarzyna Borszewska-Kornacka,3 Jozef´ Madej,2 Beata Bujak-Giz˙ycka,2 Anna Madetko-Talowska,4 Cecilie Revhaug,5 Lars O. Baumbusch,5 Ola D. Saugstad,5 Jacek Jozef´ Pietrzyk,1 and Przemko Kwinta1 1Department of Pediatrics, Jagiellonian University Medical College, Krakow, Poland 2Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland 3Neonatal and Intensive Care Department, Medical University of Warsaw, Warsaw, Poland 4Department of Medical Genetics, Jagiellonian University Medical College, Krakow, Poland 5Department of Pediatric Research, Oslo University Hospital, University of Oslo, Oslo, Norway Correspondence: Przemko Kwinta, PURPOSE. Retinopathy of prematurity (ROP) is a vision-threatening complication of a Department of Pediatrics, Jagiello- premature birth, in which the etiology still remains unclear. Importantly, the molecular nian University Medical College, processes that govern these effects can be investigated in a perturbed plasma proteome Wielicka 265, Krakow 30-663, Po- composition. Thus, plasma proteomics may add new insights into a better understanding of land; the pathogenesis of this disease. [email protected]. Submitted: June 15, 2018 METHODS. The cord and peripheral blood of neonates (30 weeks gestational age) was drawn Accepted: August 9, 2018 at birth and at the 36th postmenstrual week (PMA), respectively. Blood samples were retrospectively subdivided into ROP(þ) and ROP(À) groups, according to the development of Citation: Zasada M, Suski M, Bokiniec ROP. R, et al. An iTRAQ-based quantitative proteomic analysis of plasma proteins RESULTS. The quantitative analysis of plasma proteome at both time points revealed 30 protein in preterm newborns with retinopa- abundance changes between ROP(þ) and ROP(À) groups. After standardization to gestational thy of prematurity. Invest Ophthalmol age, children who developed ROP were characterized by an increased C3 complement Vis Sci. 2018;59:5312–5319. https:// component and fibrinogen level at both analyzed time points. doi.org/10.1167/iovs.18-24914 CONCLUSIONS. Higher levels of the complement C3 component and fibrinogen, present in the cord blood and persistent to 36 PMA, may indicate a chronic low-grade systemic inflammation and hypercoagulable state that may play a role in the development of ROP. Keywords: retinopathy of prematurity, proteome, complement, fibrinogen etinopathy of prematurity (ROP) is a biphasic ocular disease enhance our understanding of the molecular process driving R occurring primarily in infants delivered before approxi- ROP but also explain the susceptibility of some preterm mately 28 weeks gestational age with immature retina.1 It newborns to ROP. Up to now, limited research has identified includes a delayed physiologic retinal blood vessels develop- several proteins (i.e., these from the insulin-like growth factor ment, followed by anarchic vessel proliferation and a potential pathway, mitochondrial superoxide dismutase, and chordin-like for succeeding retinal detachment.2 An initial vessel loss may protein 1), in which altered levels in plasma were related to the evoke ischemic events with detrimental effects on neuronal increased risk of ROP.10,11 A study dealing with vitreous homeostasis and functions.3 Although early detection and samples of children with ROP has also revealed some proteins treatment often result in a full recovery, severe cases may lead potentially involved in the pathomechanism of ROP.12 However, to long-lasting problems of reduced visual acuity, high myopia, the limited material availability makes these studies more strabismus, nystagmus, glaucoma, late retinal detachments, or relevant in basic science but not in routine clinical practice. even blindness.4–8 Until now, the pathogenesis of ROP has not In our previous publications, we described the comparison been completely understood. The various ROP risk factors between abundances of all plasma proteins and differences at include low gestational age and low birthweight, higher or the transcriptional level (mRNA) from prematurely born fluctuating oxygen levels during oxygen therapy, hyperglyce- children with different gestational ages, both from cord blood mia, insulin use, inadequate postnatal nutrition, improper as well as at the 36th postmenstrual week (36 PMA).13–15 We insulin-like growth factor 1 concentration, poor postnatal presented that protein abundance differences are highly growth, pre- and postnatal infection, and genetic factors.9 dependent on gestation age. However, we still know too little about why some infants The goal of this study was to identify potential ROP plasma develop this complication of prematurity while others, born at biomarkers and provide a more molecular-based understanding the same age and with similar clinical characteristics, do not. of ROP by comparing a proteome profile at the two time points Studies on protein abundance profiling may not only broaden (at birth and at 36 PMA) in groups of infants with and without our knowledge about potential achievable biomarkers and help ROP. However, as our previous studies revealed, the level of Copyright 2018 The Authors iovs.arvojournals.org j ISSN: 1552-5783 5312 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/28/2021 Proteomic Analysis of Plasma Proteins in Newborns With ROP IOVS j November 2018 j Vol. 59 j No. 13 j 5313 prematurity has a profound impact on the plasma protein riched, trypsin-digested, randomly assigned to iTRAQ reagents, quantitative changes. Therefore, we performed a standardiza- labeled, and, finally, combined to corresponding 8plex tion of the obtained results to the gestational age. Our assemblies. For data normalization, each 8plex assembly secondary aim was to corroborate the protein level by a contained an internal common reference generated by complementary gene expression study. combining equal amounts of protein from all of the samples included in the measurements. Next, labeled peptides were fractionated by strong cation exchange chromatography, after METHODS which flow-through fraction and 11 consecutive injections of the elution buffer (5–500 mM ammonium acetate) were This study is an analysis of data collected as part of a multisite collected. Thus, the labeled peptides from each 8plex assembly study to investigate the transcriptome and proteome in 13–15 were distributed across 12 strong cation exchange fractions. preterm infants. The study was approved by the Jagiello- Each fraction was then separated by reversed-phase liquid nian University Bioethical Committee and adheres to the tenets chromatography and applied on-line to a Velos Pro (Thermo of the Declaration of Helsinki. Scientific, Waltham, MA, USA) mass spectrometer through a nanoelectrospray ion source. Spectra were collected in full Enrolled Patients scan mode (400–1500 Da), followed by five pairs of collisional- induced dissociation (CID) and higher energy collisional All preterm newborns with a gestational age of 30 weeks were consecutively enrolled into the study between September dissociation (HCD) tandem mass spectrometry (MS/MS) scans 1, 2013, and November 30, 2015, at the Warsaw Medical of the five most intense precursor ions from the survey full University Neonatal Intensive Care Unit (NICU), Warsaw, scan and, subsequently, merged to hybrid HCD-CID spectra by EasierMGF software.20 These were analyzed by the X!Tandem Poland. Informed consent was obtained antenatally from the 21 22 parents with an explanation of the nature and possible (The GPM Organization) and Comet search engines, consequences of the study. statistically validated with PeptideProphet, and integrated with iProphet23 under the Trans-Proteomic Pipeline (TPP) suite of software (Institute for Systems Biology, Seattle, WA, USA).24 Monitoring During Hospitalization The peptide false discovery rate (FDR) was estimated by 25 All the subjects enrolled in the study underwent careful clinical Mayu (TPP), and peptide identifications with an FDR below monitoring for symptoms of ROP, as the standard of care. The 1% were considered to be correct matches. Imputation of the 16 missing values in peptide abundances was performed in a screening of infants for ROP followed standard guidelines. 26 The examination and the qualification for laser therapy were MaxQuant environment on the log2-transformed normalized provided by an experienced specialist in ophthalmology and iTRAQ, which reports intensities with a criterion of at least retina diseases. Laser photocoagulation was performed on 75% of the values present for a peptide in the dataset by children who developed any stage ROP with plus disease in drawing the values from the normal distribution, with parameters optimized to mimic a typical low-abundance zone I, stage 3 ROP without plus disease in zone I, or stage 2 or 27 3 ROP with plus disease in zone II. In the case of ROP measurement. DanteR software was used for protein progression despite laser treatment, the patient might have quantitation and the statistical analysis of iTRAQ-labeled been qualified to receive anti-vascular endothelial growth peptides. ANOVA was performed at the peptide level by using factor therapy. a linear model with the Benjamini and Hochberg FDR correction used to adjust P values. Protein fold change