Bioanalytical solutions to characterize human proteome of clinical interest: healthy newborns and their early infections

Abstract

Early-onset sepsis (EOS), defined as a septic state manifested in the first three days of life, is one of the major causes of neonatal mortality and surviving infants are at increased risk for developing morbidities. Since the traditional methods for the diagnosis of sepsis (i.e. cultures of blood, urine, cerebrospinal fluid or bronchial fluid specimens) usually take 24 to 48 hours and clinical symptoms frequently manifest themselves in the absence of a positive culture, a faster and more unequivocal test for the differential diagnosis of infection and sepsis is needed. The main aim of the project was the investigation of saliva as a biological fluid reporting on health status of newborns, followed by the development of an innovative and noninvasive diagnostic method to differentiate bacterial from noninfectious causes of inflammation. 2-DE – MALDI-TOF/TOF and 1-DE – LC-(HR)MS/MS untargeted gel-based approaches were exploited for the characterization of the protein composition of whole saliva samples collected from healthy newborns within 48 hours after birth. The two approaches proved to be useful in providing complementary information: 2-DE was focused on the pH range 4 – 7 and most of the identified proteins have MW < 40 kDa, whereas 1-DE – LC-(HR)MS/MS analyses allowed to identify a number of proteins featuring basic pI and/or high molecular weight. As a whole, the gel-based analyses led so far, not surprisingly, to the identification of a number of proteins linked to epidermal tissue functions and, more interestingly, of a number of proteins usually highly expressed and involved in inflammatory processes, including annexin A1, glutathione S-transferase P, heat shock proteins, protein S100-A8 and -A9, probable evidence of a stressed condition especially in the early 24 hours after birth. As soon as enough samples collected from newborns affected by bacterial infection will be available, a comparison will be performed to highlight the overall alteration of physiological protein pattern as well as over- or under-expressed proteins. This will give new inputs to the development of innovative and noninvasive diagnostic methods for the early detection of EOS. In parallel with the identification of a specific target protein (or a pattern of target proteins) through the gel-based discovery approach, targeted analyses were carried out with the aim of developing an analytical method based on liquid chromatography coupled to mass spectrometry to qualitatively and quantitatively assay C-reactive protein, calcitonin and its precursor procalcitonin, acute-phase proteins widely considered plasmatic markers of sepsis, within salivary samples. Different sample preparation conditions were tested for each of the above-mentioned proteins and MALDI-TOF-based peptide mapping was exploited to compare a classical workflow of digestion in aqueous solution with the more recently described digestion in mixed organic-aqueous solution. In both cases, proteotypic peptides were identified and an optimization of the chromatographic and mass spectrometric conditions for their quantification on a Q TRAP instrument operated in MRM mode was performed. The developed methods allowed to detect the proteins of interest in the low μg/ml range. However, preliminary experiments on salivary samples showed the presence of matrix effects that need to be further investigated. Whenever possible, MSE experiments on Synapt G2-S were carried out to evaluate the applicability of a similar platform to high-throughput screening of the proteins of interest and their post-translational modifications within biological samples.