Peptide fingerprinting of folate-responsive proteins in human B lymphoblasts and orofacial clefting.

BACKGROUND

Maternal periconceptional use of folic acid contributes to the prevention of neural crest-related congenital malformations including orofacial clefts. The underlying biological pathways affected by folic acid,however, are still not clarified. In an explorative study, we identify folate-responsive proteins and pathways by advanced proteomic techniques and their possible role in orofacial development in young children.

MATERIALS AND

METHODS

At 15 months of age, we obtained B lymphoblasts from 10 children with and 10 children without an orofacial cleft. Folate-responsive protein expression was determined in folate-free B-lymphoblast cultures, supplemented with 5-methyltetrahydrofolate to reach the target concentration 30 nM. Folate-associated differences of peptide and protein expressions were assessed by analysing samples before and after folate addition. Samples were trypsin digested and measured by nano-liquid chromatography coupled online to a LTQ-Orbitrap mass spectrometer. Significantly differentiating peptides were determined using a McNemar’s test, and correlations with proteins and existing pathways were visualized using Ingenuity Pathway Analysis.

RESULTS

We found 39 folate-responsive peptides that were assigned to 30 proteins. Those proteins consisted of histones, ribosomal and heat shock proteins (HSP), and proteins involved in antioxidant reactions, cytoskeleton,glycolysis, energy production, protein processing, signal transduction and translation.

CONCLUSIONS

Histones, ribosomal and HSP were mainly found in the case group, and we confirm that almost 60% of these proteins were also found in a subset of the samples in our previous study using microarray on folate-responsive gene expression. The proteins were compared with known biological pathways and matched with recent relevant literature.