S-Palmitoylation during Retinoic Acid-Induced Neuronal Differentiation of SH-SY5Y Neuroblastoma Cells.

S-Palmitoylation is the covalent attachment of C14:0-C22:0 fatty acids (mainly C16:0 palmitate) to cysteines via thioester bonds. This lipid modification is highly abundant in neurons, where it plays a role in neuronal development and is implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The knowledge of S-palmitoylation in neurodevelopment is limited due to technological challenges in analyzing this highly hydrophobic protein modification. Here, we used two orthogonal methods, acyl-biotin exchange (ABE) and lipid metabolic labeling (LML), to identify S-palmitoylated proteins and sites during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. We identified 2002 putative S-palmitoylated proteins in total, of which 650 were found with both methods. Significant changes in the abundance of S-palmitoylated proteins were detected, in particular for several processes and protein classes that are known to be important for neuronal differentiation, which include proto-oncogene tyrosine-protein kinase receptor (RET) signal transduction, SNARE protein-mediated exocytosis, and neural cell adhesion molecules. Overall, S-palmitoylation profiling by employing ABE and LML in parallel during RA-induced differentiation of SH-SY5Y cells revealed a subset of high confidence bona fide S-palmitoylated proteins and suggested an important role for S-palmitoylation in neuronal differentiation.

Quantification of magic angle spinning dynamic nuclear polarization NMR spectra.

Dynamic nuclear polarization (DNP) allows to dramatically enhance the sensitivity of magic angle spinning nuclear magnetic resonance (MAS NMR). DNP experiments usually rely on the detection of low-γ nuclei hyperpolarized from 1H with the use of cross polarization (CP), which assures more efficient signal enhancement. However, CP is usually not quantitative. Here we determine the quantification performance of three different approaches used in MAS NMR, (conventional CP, variable contact time CP, and multiple-contact CP) under DNP conditions, and we show that absolute quantification in MAS DNP NMR is possible, with errors below 10%.