Abstract
Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β(4) and β(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.
Highlights
Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract
Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva
Data obtained from the analysis of trypsin digestion products by nano-HPLC-ESIMS/MS LTQ Orbitrap XL apparatus were elaborated by the Proteome Discoverer 1.0 program, based on SEQUEST cluster as search engine (University of Washington, USA, licensed to Thermo Electron Corp., San Jose, CA) against Swiss-Prot human proteome (March 10th, 2010 release; uniprot-taxonomy-9606-AND-reviewed-yes.fasta; 34756 nonredundant protein sequences)
Summary
Incubation was carried out at 37 °C and after 40 min the solution was centrifuged at 8000 ϫ g for 5 min, and the supernatant immediately analyzed by RP-HPLC-ESI-MS. Removal of N-Terminal Acetylation—Freeze-dried powder of the purified protein or of protein mixtures was incubated in the presence of 25% TFA at 55 °C for 1h and the solution was analyzed by RPHPLC ESI-MS. In the determination of XIC peak area a correct choice of the m/z values for protein detection is necessary, avoiding m/z potentially overlapping with ESI spectra of other closeeluting proteins in crowded chromatographic elution ranges (see “Results”). Data obtained from the analysis of trypsin digestion products by nano-HPLC-ESIMS/MS LTQ Orbitrap XL apparatus were elaborated by the Proteome Discoverer 1.0 program, based on SEQUEST cluster as search engine (University of Washington, USA, licensed to Thermo Electron Corp., San Jose, CA) against Swiss-Prot human proteome (March 10th, 2010 release; uniprot-taxonomy-9606-AND-reviewed-yes.fasta; 34756 nonredundant protein sequences). Precursor mass search tolerance was set to 10 ppm and fragment mass tolerance was set to 1.5 Da
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