Protein glycosylation is an essential post-translational modification boosting proteomic diversity. The major groups of human plasma glycoproteins are immunoglobulins (Igs) and acute phase proteins (APPs). Changes in plasma Igs and APPs levels and their glycosylation have been previously associated with inflammatory diseases, several types of cancer, pregnancy, or aging. In this study, we present a novel approach to investigate the neonatal and maternal plasma at the end of pregnancy using a combination of targeted quantitative proteomics and total plasma N-glycome profiling. We present a new SRM-based multiplex assay to quantify 19 APPs and 7 Igs. We applied this assay to 98 samples of umbilical cord plasma and corresponding maternal plasma samples and observed significantly lower levels of all analyzed proteins in neonates, apart from the glycoprotein alpha-2-macroglobulin. Unlike previous methods, our SRM-MS assay allows us to quantify dozens of proteins simultaneously and to distinguish protein variants. We compared maternal and fetal total plasma-released N-glycomes in the same set of samples. We quantified 66 individual glycans, which were used to calculate 73 glycosylation traits. In neonates, the major N-glycans were di-antennary complex-type with core fucosylation, and the production of higher, branched, sialylated N-glycans was limited.

The plasma sample was diluted 50-fold using 50 mM ammonium bicarbonate buffer (ABB), and 30 µl of the diluted plasma sample was transferred into a 96-well plate. The sample was reduced (3 µl of 200 mM DTT, 95°C, 10 min), then cooled down to ambient temperature, and cysteine residues were alkylated (3 µl of 400 mM IAA, ambient temperature, 30 min in the dark). Trypsin was added to the sample in a 1:40 ratio (w/w of the enzyme to total protein content) for enzymatic proteolysis (37°C, 16 h). The proteolysis was quenched by adding 200 µl of 2% FA in water (pH < 3). Working solutions containing the mixture of SIL peptide internal standards were added (2.5 µl), and the final concentration of each SIL peptide in the sample was 150 nM. Tryptic peptides were desalted using a reverse-phase solid phase extraction (SPE) cartridge in a 96-well plate format (Oasis PRIME HLB 30mg, Waters, Milford, MA). The sample was loaded onto the cartridge, washed with 300 µl water with 2% FA, pH < 3, and eluted twice with 200 µl of 50% ACN in water with 2% FA, pH < 3. The extract was dried in a vacuum concentrator centrifuge (Savant SPD121 P SpeedVac, Thermo Fisher). The sample was reconstituted in 25 µl of 5% ACN in water with 0.1% FA before UHPLC-SRM analysis. Samples were analyzed using the UHPLC system (InfinityTM 1260 Agilent Technologies, Santa Clara, CA) and a reversed-phase analytical column (C18 Peptide CSH; 1.7 µm, 2.1 mm i.d. x 100 mm; cat. #186006937; Waters; Milford, MA). The sample injection volume was 3 µl. The mobile phase flow rate was 0.3 ml/min with mobile phases A (0.1% FA in water) and B (0.1% FA in 95% ACN) in the linear gradient elution mode (0-25 min) with a wash step (25.30-30 min) and re-equilibration step (31-35 min). The gradient program was as follows: 0.0 min 5% B; 25 min 30% B; 25.3 min 95% B; 30 min 95% B; 31 min 5% B; 35 min 5% B. Column temperature was 40°C and autosampler temperature was 5°C. SRM protein assays were performed using a triple quadrupole mass spectrometer (Agilent 6495B, Agilent Technologies, USA) with a standard-flow Jet Stream electrospray ionization (ESI) source. ESI parameters were as follows: capillary voltage 3.5 kV, nozzle voltage 800 V, gas flow rate 20 l/min at 220°C, sheath gas pressure 25 PSI and flow rate 10 l/min at 400°C. Positively charged ions were detected in dynamic SRM mode with 2 min retention time window centered on the peptide experimental retention time. All tryptic peptides and SIL peptide internal standards were identified using 3 SRM transitions (qualifiers) and quantified using a single quantifier SRM transition. In total, 334 transitions were monitored with a total cycle time of 500 ms.

Study samples and data were obtained from the Central European Longitudinal Studies of Parents and Children: The Next Generation (CELSPAC: TNG).The CELSPAC: TNG study was approved by the Multicentre and Local Ethical Committee of University Hospital Brno, the Czech Republic (No. 20140409-01, date 2014/04/09). In total, 98 samples of UCP and 98 corresponding samples of MP were used in this study. The cohort study included only singleton pregnancies. MP samples were collected between the 38th and 41st week of pregnancy, and UCP samples were collected immediately after delivery. All plasma samples were immediately placed at -80˚C and stored until sample processing and analysis. The newborn study subjects were female (n=46) and male (n=52) neonates. The gestation age [week + days] ranged from 38+2 to 41+3. The average birth weight was 3510 g, ranging from 2470 to 4560 g. Ten neonates were delivered by Cesarean section.