Targeted LC-MS/MS for the evaluation of proteomics biomarkers in the blood of neonates with necrotizing enterocolitis and late-onset sepsis.

Late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) are severe life-threatening conditions for neonates. Accurate, early diagnosis and timely initiation of treatment are crucial. Non-specific overlapping clinical signs along with the non-sensitive/specific diagnostic tools set obstacles to speedy, trustful diagnosis including differential diagnosis. The objective of this study was to evaluate the potential of targeted LC-MS/MS proteomics in identifying diagnostic biomarkers of NEC or LOS. We conducted a prospective case-control study evaluating serum proteomics profiles of 25 NEC, 18 LOS, and an equal number of matched control neonates, over three sampling points. Eighty-three concatemers and synthetic peptides belonging to 47 protein markers of the two diseases were selected after thorough literature search. A novel selected reaction monitoring (SRM), LC-MS/MS method was developed for their analysis and evaluation as potential biomarkers. Multivariate and univariate statistical analyses highlighted significant proteins in differentiating LOS and NEC neonates and diseased from controls. Moreover, panels of proteins were tested for their ability to distinguish LOS from NEC and controls. We suggest two panels of three proteins each, exhibiting very high diagnostic value for LOS and excellent diagnostic performance at the critical LOS-NEC differentiation, reaching an AUC ROC value close to 1 (0.999). These panels constitute a valuable starting point for further validation with broader cohorts of neonates, aiming to improve the clinical practice. Graphical abstract ᅟ.

Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9).

The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a homodimeric complex, which translocates peptides across the membrane. Peptide transport strictly requires ATP hydrolysis. The transport follows Michaelis-Menten kinetics with low affinity and high capacity. Different nucleotides bind and energize the transport with a slight predilection for purine bases. The peptide specificity is very broad, ranging from 6-mer up to at least 59-mer peptides with a preference for 23-mers. Peptides are recognized via their backbone, including the free N and C termini as well as side chain interactions. Although related to TAP, TAPL is unique as far as its interaction partners, transport properties, and substrate specificities are concerned, thus excluding that TAPL is part of the peptide-loading complex in the classic route of antigen processing via major histocompatibility complex class I molecules.