The use of proteomics for blood biomarker research in premature infants: a scoping review

Over the last decade, the use of proteomics in the setting of prematurity has increased and has enabled researchers to successfully identify biomarkers for an array of associated morbidities. The objective of this scoping review was to identify the existing literature, as well as any knowledge gaps related to proteomic biomarker discoveries in the setting of prematurity. A scoping review was conducted using PubMed, Embase and Medline databases following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines. The study selection process yielded a total of 700 records, of which 13 studies were included in this review. Most studies used a tandem Mass Spectrometry (MS/MS) proteomics approach to identify key biomarkers. The corresponding studies identified proteins associated with retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), necrotising enterocolitis (NEC), late onset sepsis (LOS) and gestational age. This scoping review demonstrates the limited use of proteomics to identify biomarkers associated with severe complications of prematurity. Further research is warranted to identify biomarkers of other important morbidities associated with prematurity, such as intraventricular haemorrhage (IVH) and cerebral palsy, and to investigate the mechanisms associated with these outcomes.


Introduction
Proteomics is a methodological approach that allows for the analysis of many proteins simultaneously and has been successful in identifying many novel disease biomarkers [1]. Proteomic methodologies have been previously used in varying contexts, such as discovering biomarkers of diabetic nephropathy and identifying novel diagnostic markers of cancer [2,3]. Plasma proteomics is advantageous as it only uses a small volume of blood to study hundreds and sometimes thousands of proteins, and can identify changes in protein expression that may occur with age and disease [4]. Proteomics is not limited to analysis of blood samples, and enables the use of biological fluids such as saliva and urine, and tissue samples (e.g. tumours) [5]. Due to the small volume required for analysis, plasma proteomics has become increasingly popular and has enabled investigations of plasma proteins in vulnerable populations such as in paediatrics, as well as in critically ill patients, where blood may be scarce and not readily available for research purposes [4].
Preterm birth is the leading cause of death among the paediatric population globally [6]. With major technological advances in neonatal care over the last few decades, there has been an increase in survival of infants born preterm (< 37 weeks' gestation), in particular those born extremely preterm (< 28 weeks' gestation) [7]. Despite the technological advances that have improved survival in these vulnerable populations, preterm birth is associated with significant morbidities including intraventricular
Within the last decade proteomics has enabled researchers to identify predictive biomarkers of NEC in preterm infants using buccal swabs [9]. More specifically, plasma proteomics has previously identified proteins that may play a role in the development of retinopathy of prematurity [10]. However, to date there has been limited research into plasma protein biomarkers in predicting other outcomes in preterm infants. Consequently, a scoping review was conducted to understand the current state of knowledge in this space, and to identify knowledge gaps that could be addressed by future studies. A preliminary search of MEDLINE, PubMed, JBI Evidence Synthesis and Embase was conducted and did not identify any current systematic reviews or scoping reviews on this topic. Thus, this review is novel and will make a significant contribution to the understanding and knowledge in the use of proteomics in preterm infants.

Review question
The following research question was formulated using the PCC (Population, Concept, Context) framework: What is the existing proteomic evidence of blood biomarker research in the setting of prematurity?

Study design
This scoping review was conducted based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist [11].

Search strategy
The following three electronic databases: Medline, Embase and PubMed were searched on the 24th September 2020 for all peer-reviewed studies. An additional search for grey literature was conducted using the Open-Grey and GreyLit databases. The specific search terms used for each database are detailed in Appendix A. In summary, studies included in this review were identified using the search terms ['preterm' OR 'premature') AND ['proteome' OR 'protein-analysis'] AND ['blood-protein' OR [biomarker'], as well as including derivatives of these terms. Studies identified in this review were limited to those written in the English language and conducted in humans only. Studies retrieved using these search terms and parameters were screened by two authors (NL and TC), initially focusing on the eligibility of the studies' titles and abstracts using the following inclusion and exclusion criteria.
Exclusion criteria: (I) infants born at term or postterm (≥ 37 weeks), (II) proteome of other biological samples (e.g. saliva or urine) assessed, (III) case report, review, conference abstract or editorial correspondence and (IV) animal studies.

Data extraction and charting
Studies that were chosen for full-text assessment were assessed by NL and TC and with any discrepancies and uncertainties, a third reviewer (VI) was to assess the studies. Data extracted included publication year, disease/outcome assessed, aims, study population, comparative groups, proteomic methodology, proteinpathway analysis, key findings and study limitations. The detailed assessment for each critically reviewed study is presented in Table 1.

Results
The initial search identified 678 studies using the scoping review search strategy, with an additional 22 studies identified using the grey literature search. After the removal of duplicates, 462 publications remained for title and abstract screening. A vast majority of studies (n = 444, 96%) were excluded due to not fulfilling the inclusion criteria or having no relevance to the topic of prematurity and blood biomarker discoveries. Eighteen studies underwent full-text review, with three studies excluded because they did not primarily investigate biomarkers of disease and outcomes. One study of children born preterm did not collect samples at birth and one study presented data in brief report, which did not include any proteomic data. Figure 1 illustrates the article screening and selection process, following the PRISMA guidelines (Fig. 2).

Description of included studies
A total of thirteen studies met the inclusion criteria for this scoping review and are summarised in Table 1. Eleven of the thirteen included studies investigated proteins and their associations with known outcomes of prematurity. The participant gestational age at birth ranged from < 23 to 37 weeks, with sample sizes varying from 4 to 77 participants. Most studies used a tandem Mass Spectrometry method (MS/MS) to analyse the  proteins of interest [10,[12][13][14][15]. Three of the fourteen studies also conducted protein validation and completed this task using protein microarray and immunoassay techniques [10,16,17]. Approximately half of the studies (n = 7, 47%) were completed using plasma samples (Fig. 3). The proteins identified as proteins of interest across the 13 studies included in this scoping review, with reference to the specific study/ies are summarised in Table 2.

Retinopathy of prematurity (ROP)
Two studies investigated the outcome associated with prematurity, ROP [10,18]. ROP is seen most commonly among infants born very preterm (< 32 weeks' gestational age) or < 1250 g birth weight. Abnormal blood vessel development occurs in the retina in response to oxygen exposure, which can lead to retinal detachment and blindness in severe cases [18]. Currently there is no existing method to predict the occurrence of ROP in infants born preterm or born with a low birth weight and all high-risk infants are routinely screened. Hence, a proteomic approach was adopted to identify underlying biomarkers of the disease [10,18]. Several biomarkers of the complement and inflammatory system were identified in infants who developed ROP [10]. Lynch et al. identified mitochondrial Superoxide dismutase (MnSOD), an antioxidant located in the mitochondria, as a potential therapeutic target for significant ROP [18].

Bronchopulmonary dysplasia (BPD) and pulmonary vascular disease (PVD)
Two of the thirteen included studies investigated plasma proteins and their association with BPD [15,19]. BPD is a chronic lung disease that affects infants born preterm [20]. Arjaans et al. implemented the use of a SOMAscan proteomic assay, whereas Zasada et al. utilised MS/MS approach to identify key biomarkers of BPD. Both studies identified several proteins that may be used in future diagnosis of BPD as well associations between severity and disease prognosis [15,19]. Wagner et al. investigated plasma proteins and their association with the pathogenesis of PVD, a term used to describe abnormal function and vascular growth of the lungs. They identified 18 proteins that were associated with PVD, including proteins associated with growth factors, angiogenesis and the extracellular matrix [21]. The protein analysis conducted by Wanger et al. also identified proteins of several different biological process pathways (e.g. Tissue Inhibitor of Metalloproteinases 3 (TIMP-3) used in platelet degradation and Bone proteoglycan II, involved in degradation of the extracellular matrix (ECM)) that may be associated with PVD.

Necrotising enterocolitis (NEC) and late onset sepsis (LOS)
Two of the thirteen studies examined biomarkers for NEC and LOS [12,16]. Ng [12]. Interestingly, the only protein common across the two studies was SAA [12,16].

Gestational age and signalling pathways
Suski et al. completed several studies [13,14] investigating plasma proteome changes in preterm infants comparing gestational ages [13] and malfunctioning proteins in various signalling pathways [14]. Utilising a tandem MS approach they were able to identify proteomic changes across varying gestational ages for several pathways which include; coagulation, inflammation, complement activations and immunomodulation [13,14]. Suski et al. also observed Complement C3, Factor V and Complement C4-A were associated with gestational age [13]. LRG1 was the only common protein identified across the two studies [13,14].

Discussion
In this scoping review we identified 13 primary studies that used proteomics to identify blood protein biomarkers in the setting of prematurity that used either plasma or serum as the sample which was analysed. It is important to note that studies conducted in serum cannot be directly compared to studies conducted in plasma as these are two entirely different samples. Unlike plasma which is prepared only via centrifugation, Preparation of serum entails formation and removal of a blood clot   [18,19] Macrophage-stimulating protein receptor MST1R Q04912 ATP/ enzyme binding Cell migration/ hepatocyte growth factor receptor signalling pathway [18,21] Glycoprotein hormones alpha chain CGA P01215 Follicle-stimulating hormone activity Peptide hormone processing [25] Cystatin-M CST6 Q15828 Cysteine-Type Endopeptidase Inhibitor Activity Anatomical structure morphogenesis [18] Plasminogen PLG P00747 Apolipoprotein Binding/ protein domain specific binding Blood coagulation/ cellular protein metabolic process [18] Insulin-like growth factor-binding protein 7

IGFBP-7 Q16270
Insulin-Like Growth Factor Binding Cell Adhesion/ cellular protein metabolic process [18] Plasma protease C1 inhibitor SERPING1 P05155 Serine-Type Endopeptidase Inhibitor Activity Blood coagulation, intrinsic pathway/ complement activation, classical pathway [13] Complement C3 C3 P01024 C5L2 anaphylatoxin chemotactic receptor binding Cellular protein metabolic process [10,13] Coagulation factor V F5 P12259 Copper ion binding Cellular protein metabolic process/ platelet degranulation [13] Complement C4-A C4A P0C0L4 Endopeptidase inhibitor activity Cellular protein metabolic process/ regulation of complement activation [13] Leucine-rich alpha-2-glycoprotein LRG1 P02750 Transforming growth factor beta receptor binding Neutrophil degranulation [13,14]  Angiogenesis/ leukocyte migration [19] Angiopoietin-1 ANGPT1 Q15389 Receptor tyrosine kinase binding Angiogenesis/ leukocyte migration [19] Bone morphogenetic protein 10 BMP10 O95393 Growth factor/ cytokine activity Cell adhesion/ BMP signalling [19] Hepatocyte growth factor receptor MET P08581 ATP binding/ protein tyrosine kinase activity cell surface receptor signalling pathway/ cell migration [19] Protein S100-A12 S100A12 P80511 Calcium/ion binding Cytokine secretion/ inflammatory response [24] Interleukin-6 IL6 P05231 Cytokine/ growth factor activity Cellular protein metabolic process/ acute-phase response [24] Interleukin-10 IL10 P22301 Cytokine/ growth factor activity B cell differentiation/ cytokine-mediated signalling pathway [24] Complement receptor type 2 CR2 P20023 Complement binding/ DNA binding B cell differentiation/ immune response [25] Coagulation factor VII F7 P08709 Calcium ion binding/ signalling receptor binding Blood coagulation-extrinsic pathway [25] Coagulation factor XI F11 P03951 Heparin binding Blood coagulation-intrinsic pathway/ plasminogen activation [25] L-selectin SELL P14151 Calcium ion binding Leukocyte migration/ regulation of immune response [25] Interleukin-2 receptor subunit alpha IL2RA P01589 Interleukin-2 binding/ receptor activity cytokine-mediated signalling pathway [25] Platelet glycoprotein VI GP6 Q9HCN6 Collagen binding/ signalling receptor activity Blood coagulation/ platelet activation/ leukocyte migration [25] Collectin-12 COLEC12 Q5KU26 Galactose binding/ low-density lipoprotein particle binding Receptor-mediated endocytosis/ regulation of immune response [25] Follistatin-related protein 3 FSTL3 O95633 Activin/ fibronectin binding Cellular protein metabolic process/ cell differentiation [25] Growth/differentiation factor 15 GDF15 Q99988 BMP receptor binding/ growth factor activity Activation of MAPK activity/ BMP signalling [25] Insulin-like growth factor-binding protein 1 IGFBP1 P08833 Insulin-like growth factor binding Cellular protein metabolic process [25] activating not only coagulation proteins but also changing the concentration of inflammatory proteins, a scenario that reflects the manipulation itself and not the physiological setting. Similarly, a cord-blood sample is different to the blood sample collected from babies at birth, due to differences in the vasculature of the umbilical cord and blood vessels within the newborn. Our findings indicate that the focus of research in the setting of blood protein biomarkers in the setting of prematurity focused on several diseases, such as ROP, BPD, LOS and NEC. However, there has been a lack of research focusing into other outcomes known to be associated with preterm birth such as cerebral palsy, intraventricular haemorrhage, or hypertension. To our best knowledge, none of the findings from the studies included in our scoping review have been translated into the clinical setting. Blood proteomic studies within this population may reflect a lack of collaboration between clinicians and proteomic experts, as well as difficulty in accessing samples from premature babies, factors that could be overcome, particularly in research institutes associated with tertiary hospitals [22].

Limitations of current published studies
The main limitation of the studies included in this review are the small sample sizes represented in those studies. Future studies should be adequately powered, and a shift of the primary focus from not only understanding mechanism of disease, but also on identifying proteins that are associated with outcomes or disease and which can be used in the clinical setting to improve outcomes for premature infants.

Conclusions
This scoping review identified a paucity of evidence around biomarker discoveries in the population of preterm infants. Several proteomic methods, including tandem mass spectrometry, immunoassays, and MALDI-TOF MS, have been used to identify biomarkers for various outcomes (e.g. ROP and BPD) associated with preterm birth. This review identifies the need for future research focusing on biomarkers to understand the possible mechanisms related to preterm birth, as well as to identify predictive protein biomarkers for complications or long-term sequelae associated with preterm birth, such as intraventricular haemorrhage and hypertension.