Label-free quantitative proteomic analysis of extracellular vesicles released from fibroblasts derived from patients with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that represents a significant cause of infant mortality. SMA is characterized by reduced levels of the Survival Motor Neuron protein leading to the loss of alpha motor neurons in the spinal cord and brain stem as well as defects in peripheral tissues such as skeletal muscle and liver. With progress in promising therapies such as antisense oligonucleotide and gene replacement, there remains a need to better understand disease subtypes and develop biomarkers for improved diagnostics and therapeutic monitoring. In this study, we have examined the utility of extracellular vesicles as a source of biomarker discovery in patient-derived fibroblast cells. Proteome examination utilizing data-independent acquisition and ion mobility mass spectrometry identified 684 protein groups present in all biological replicates tested. Label-free quantitative analysis identified 116 statistically significant protein alterations compared to control cells, including several known SMA biomarkers. Protein level differences were also observed in regulators of Wnt signaling and Cajal bodies. Finally, levels of insulin growth factor binding protein-3 were validated as being significantly higher in extracellular vesicles isolated from SMA cells. We conclude that extracellular vesicles represent a promising source for SMA biomarker discovery as well as a relevant constituent for advancing our understanding of SMA pathophysiology.

Characterization of Phosphopeptide Positional Isomers on the Transcriptional Co-activator TAZ.

The transcriptional co-activator with the PDZ binding motif (TAZ) is a critical regulator of numerous cellular processes such as cell differentiation, development, proliferation, and cell growth. Aberrant expression and activity of TAZ are also featured in many human malignancies. A hallmark of TAZ biology is its cytoplasmic retention mediated by 14-3-3 isoforms in response to phosphorylation of Ser89 by members of the LATS family of kinases. Following the observation that TAZ is a highly phosphorylated protein even when Ser89 is mutated, high-resolution mass spectrometry employing data-independent acquisition and ion mobility separation was conducted to elucidate additional TAZ phosphorylation sites that may play a role in regulating this critical transcriptional rheostat. Numerous phosphorylation sites on TAZ were identified, including several novel modifications. Of notable interest was the identification of positional phosphoisomers on a phosphopeptide containing Ser89. Optimized use of a so-called wideband enhancement acquisition technique yielded higher-quality fragmentation data that confirmed the detection of Ser93 as the positional phosphoisomer partner of Ser89 and identified diagnostic fragment ions for the phosphorylation events. Functional analysis indicated that Ser93 phosphorylation reduces the level of 14-3-3 association and increases the level of nuclear translocation, indicating this phosphorylation event attenuates the 14-3-3-mediated TAZ cytoplasmic retention mechanism. These findings suggest that the biological activities of TAZ are likely dynamically regulated by multisite phosphorylation.