Integrated Proteomic HPLC-ESI-MS Analysis of Salivary Peptides of Preterm Newborns: a Pilot Study

Background

The study of human fetal development is hampered by the difficulty to have an experimental plan and a specimen collection ethically acceptable. In the past years the analysis of saliva of preterm newborns is demonstrated to be a powerful tool to investigate human fetal development in an ethically acceptable fashion [1]. The collection of salivary samples is indeed a non-invasive procedure which is usually performed by the personnel of the Neonatology Unity for the wellbeing and survival of highly preterm newborns. The samples collected are usually discharged, while their analysis, after the informed consent of one of the parents, could be precious to investigate molecular events occurring during the last months of fetal life.

Indeed, previous HPLC-ESI-MS analyses carried out on salivary samples collected from newborns at different postmenstrual age (PMA) evidenced the presence of high levels of more than 40 proteins, many of them pertaining to the family of S100 proteins, decreasing their amounts according to the PMA [1]. Commonly, these proteins are either absent or present at a concentration near to the limit of sensitivity of our MS apparatus at the normal term of delivery and in adult saliva. In the first years of life these proteins are slowly replaced by the protein present in the human adult saliva [2]. The first adult salivary proteins appearing in the preterm newborn at 180-190 days of PMA are the acidic proline-rich proteins (aPRPs) isoforms encoded by the PRH2 locus, together with a glycosylated basic proline-rich protein expressed by the PRB3 locus, followed at 210-220 days of PCA by histatins, statherin, and P-B peptide. Acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared after 4 years (±1 year) of age and reach the level observed in the adult after the puberty [2]. These protein families specific of human adult saliva are differently secreted by the major salivary glands (parotid, submandibular and sublingual) and during the secretion process are submitted to several post-translational modifications (PTMs) under the action of various enzymes (i.e kinases and proteinases) common to other exocrine and endocrine organs [3-6]. Studies carried out by our research group showed for instance that the activity of Fam20C, a pleitropic Golgi kinase involved in the phosphorylation of many different substrates in many organs and tissues [7], responsible for the phosphorylation of aPRPs, histatin 1, statherin and S-type (salivary) cystatins is not active at 180 days of PMA. It slowly increases its activity reaching values comparable to adults at about 2 years of age [2,8]. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns of 180 days of PMA [2]. These studies suggested also that the proteinases involved in the proteolysis of salivary proteins were more active in preterm newborns.

Aim of the study

The aim of the study will be to establish the structure and the relative amount over time of several peptides and proteins detectable in preterm saliva still pending for a precise characterization, with particular regard to enzymes with proteolytic or oxidative activity. Oxidative stress and proteolysis are well known pathogenetic mechanisms involved in pathological conditions of preterm infants such as bronchopulmonary dysplasia and retinopathy of prematurity [9-11]. A correlation of the different expression over time of the enzymes identified with the prevalence of these pathological conditions can be also evaluated and represent the basis for future studies aim to verify the utility of such peptides as pathological markers. The proteomic approach utilized will be a targeted ESI mass spectrometry, based on a top-down platform devoted to the analysis of the intact salivary proteome.