Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation.

Understanding the role of DNA damage checkpoint kinases in the cellular response to genotoxic stress requires the knowledge of their substrates. Here, we report the use of quantitative phosphoproteomics to identify in vivo kinase substrates of the yeast DNA damage checkpoint kinases Mec1, Tel1, and Rad53 (orthologs of human ATR, ATM, and CHK2, respectively). By analyzing 2,689 phosphorylation sites in wild-type and various kinase-null cells, 62 phosphorylation sites from 55 proteins were found to be controlled by the DNA damage checkpoint. Examination of the dependency of each phosphorylation on Mec1 and Tel1 or Rad53, combined with sequence and biochemical analysis, revealed that many of the identified targets are likely direct substrates of these kinases. In addition to several known targets, 50 previously undescribed targets of the DNA damage checkpoint were identified, suggesting that a wide range of cellular processes is likely regulated by Mec1, Tel1, and Rad53. mass spectrometry ͉ Mec1 ͉ N-isotag ͉ phosphorylation ͉ Rad53

Dominant mutations in two functionally related DNA/RNA-binding proteins, trans-activating response region (TAR) DNA-binding protein with a molecular mass of 43 KDa (TDP-43) and fused in sarcoma/ translocation in liposarcoma (FUS/TLS), cause an inherited form of ALS that is accompanied by nuclear and cytoplasmic aggregates containing TDP-43 or FUS/TLS. Using isogenic cell lines expressing wild-type or ALS-linked TDP-43 mutants and fibroblasts from a human patient, pulse-chase radiolabeling of newly synthesized proteins is used to determine, surprisingly, that ALS-linked TDP-43 mutant polypeptides are more stable than wild-type TDP-43. Tandem-affinity purification and quantitative mass spectrometry are used to identify TDP-43 complexes not only with heterogeneous nuclear ribonucleoproteins family proteins, as expected, but also with components of Drosha microprocessor complexes, consistent with roles for TDP-43 in both mRNA processing and microRNA biogenesis. A fraction of TDP-43 is shown to be complexed with FUS/TLS, an interaction substantially enhanced by TDP-43 mutants. Taken together, abnormal stability of mutant TDP-43 and its enhanced binding to normal FUS/TLS imply a convergence of pathogenic pathways from mutant TDP-43 and FUS/TLS in ALS.mass spectrometry | protein stability | amyotrophic lateral sclerosis | microRNA | ribonucleoproteins

The RNA-binding protein FUS/TLS, mutation in which is causative of the fatal motor neuron disease ALS, is demonstrated to directly bind to the U1-snRNP and SMN complexes. ALS-causative mutations in FUS/TLS are shown to abnormally enhance their interaction with SMN and dysregulate its function, including loss of Gems and altered levels of small nuclear RNAs (snRNAs). The same mutants are found to have reduced association with U1-snRNP. Correspondingly, global RNA analysis reveals a mutant-dependent loss of splicing activity, with ALS-linked mutants failing to reverse changes caused by loss of wild-type FUS/TLS. Furthermore, a common FUS/TLS mutant-associated RNA splicing signature is identified in ALS patient fibroblasts. Taken together, these studies establish potentially converging disease mechanisms in ALS and spinal muscular atrophy, with ALS-causative mutants acquiring properties representing both gain (dysregulation of SMN) and loss (reduced RNA processing mediated by U1-snRNP) of function.