In-utero and dietary factors make important contributions towards health and development in early childhood. In this respect, serum proteomics of maturing infants can provide insights into studies of childhood diseases, which together with perinatal proteomes could reveal further perspectives. Accordingly, to determine differences between feeding groups and changes in infancy, serum proteomics analyses of mother-infant dyads with HLA-conferred susceptibility to type 1 diabetes (n = 22), weaned to either an extensively hydrolyzed or regular cow’s milk formula, were made. The LC-MS/MS analyses included samples from the beginning of third trimester, the time of delivery, 3 months postpartum, cord blood and samples from the infants at 3, 6, 9 and 12 months. Correlations between ranked protein intensities were detected within the dyads, together with perinatal and age-related changes. Comparison with intestinal permeability data revealed a number of significant correlations, which could merit further consideration in this context.

Selected reaction monitoring (SRM) mass spectrometry was used to verify the relative abundance of the targets GGH, PON3, FCN3, PGLYRP2, CERU, MASP1 and the immunoglobulins KVD30, KV240 and LV403, in the serum samples. A1GB, ALB were also measured as reference proteins, and fibrinogen and HBB as quality measures for potential plasma contamination/selection and hemolysis, respectively. Heavy-labeled synthetic peptides (lysine 13C6 15N2 and arginine 13C6 15N4) peptides were obtained for the targets of interest (PEPotec, Grade 2, Thermo Fischer Scientific). The targets were selected on the basis of the detection of their differential expression in the data-dependent LC-MS/MS measurements. Skyline software was used to create the scheduled method for data acquisition and process the data, i.e. to check the peak assignments and their integration. The trypsin digested samples from both the discovery and follow-up analyses were spiked with synthetic heavy labelled analogues of the peptide targets and a retention time standards (MSRT1, Sigma) for scheduled selected reaction monitoring. The LC-MS/MS analyses were conducted using an Easy-nLC 1000 liquid chromatograph (Thermo Scientific) coupled to a TSQ Vantage Triple Quadrupole Mass Spectrometer (Thermo Scientific). The column configuration included a 20 x 0.1 mm i.d. pre-column in conjunction with a 150 mm x 75 µm i.d. analytical column, both packed with 5 µm Reprosil C18-bonded silica (Dr Maisch GmbH). The following separation gradient was employed at a flow rate of 300 nl/min; from 5% to 21% B in 11 min, then to 36% B in 9 min, to 100% in 2 min, then ending with an 8 min isocratic period. The mobile phase compositions were the same as those used for the DDA analysis. The estimated injected amounts was 250 ng of endogenous sample, spiked with 50 fmol of synthetic peptides.

Infant participants were recruited from pregnant mothers either with a HLA defined T1D-risk genotype, or with a partner with a HLA defined T1D-risk. The T1D risk of the offspring was assessed by HLA genotyping from cord blood. Serum samples were collected at 9 months from birth from subjects in regular and extensively hydrolyzed cow’s milk-based formula groups, 26 and 36 subjects, respectively.