Top-down proteomics for the investigation of saliva during human development

Aim of this study was to evaluate by a label-free top-down proteomic platformsvariations during human development of the level of different proteins detectable in adult saliva and their post-translational modifications (PTMs). The relative amounts of different proteoforms of acidic-proline-rich proteins (aPRPs), basic proline-rich proteins, statherin, histatins 1, 3, 5 and 6, cystatin S, S1, S2, SA and SN and S100A9 were measured in 312 samples (111 serially collected from 17 preterm newborns at different post-conceptional age (PCA), 178 from subjects aged between 0 and 18 years, and 23 from adults). The different proteoforms of these protein families were expressed according to this order: aPRPs(PRH2 locus[1]) and g-PRP(PRB3 locus)before 195 days of PCA; histatin 1 and statherin at 210-220 days of PCA; aPRPs (PRH1 locus)at normal term of delivery (270-280 days of PCA); cystatin S proteoforms after normal term of delivery;bPRPs (PRB1, PRB2 and PRB4 loci) later, reaching adult levels after puberty [2].All S100A9 proteoformswere highly abundant in pre-term newborn saliva, their concentration decreasing as a function of PCA [3]. The PTMsevaluation of various proteoforms highlighted that the Golgi casein kinase (Fam20C [4]), responsible for the aPRPs, histatin 1, statherin and cystatin Sphosphorylation, is poorly active during fetal development. The p38 mitogen-activated protein kinase 14(MAPK14) responsible for S100A9 phosphorylation, the convertases, thecarboxy-peptidases, themethionine aminopeptidasesand N-terminal-acetyl-transferasesresponsible for the cleavage and processing of different proteins (aPRPs, bPRPs, histatins, S100A9) during secretionare fully active before 195 days of PCA.