A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. RESULTS: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. CONCLUSIONS: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

Targeted analysis of tyrosine phosphorylation by immuno-affinity enrichment of tyrosine phosphorylated peptides prior to mass spectrometric analysis.

Tyrosine phosphorylation is a key process that regulates seminal biological functions, hence, deregulation of this mechanism is an underlying cause of several diseases including cancer and immunological disorders. Due to its low abundance, tyrosine phosphorylation is typically under-represented in most of the global MS-based phosphoproteomic studies. Here, we describe a selective approach based on immuno-affinity purification using specific antibodies to enrich tyrosine phosphorylated peptides from a complex proteolytic digest. LC-MS/MS analysis is subsequently used for peptide identification allowing the exact localization of the phosphorylated residue within the sequence. Using this approach more than 1000 non-redundant phosphotyrosine peptides can be identified in less than 6h of MS analysis, reflecting the high sensitivity and specificity of the technique. The identified tyrosine phosphorylated peptides can be used to study different biological aspects of tyrosine signaling and disease.

Investigating the role of FGF-2 in stem cell maintenance by global phosphoproteomics profiling.

Human embryonic stem cells (hESCs) are of immense interest for regenerative medicine as a source of tissue replacement. Expansion of hESCs as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. One of the key growth factors that maintains this balance is fibroblast growth factor-2 (FGF-2). However, the underlying molecular mechanisms are poorly understood. We recently profiled specifically tyrosine phosphorylation events that occur during FGF-2 stimulation of hESCs (Ding et al., J. Cell. Physiol. 2010, 225, 417-428). Here, we complement this phosphoproteome profiling by analyzing temporal dynamics of mostly serine and threonine protein phosphorylation events. Our multi-dimensional strategy combines strong cation exchange chromatography to reduce the sample complexity followed by titanium dioxide off-line for the enrichment of phosphopeptides and dimethylation-based stable isotope labeling for quantification. This approach allowed us to identify and quantify 3261 unique proteins from which 1064 proteins were found to be phosphorylated in one or more residues (representing 1653 unique phosphopeptides). Approximately 40% of the proteins (553 unique phosphopeptides) showed differential phosphorylation upon FGF-2 treatment. Among those are several members of the canonical pathways involved in pluripotency and self-renewal (e.g. Wnt and PI3K/AKT), hESC-associated proteins such as SOX2, RIF1, SALL4, DPPA4, DNMT3B and 53 proteins that are target genes of the pluripotency transcription factors SOX2, OCT4 and NANOG. These findings complement existing pluripotency analyses and provide new insights into how FGF-2 assists in maintaining the undifferentiated state of hESCs.

Finding the same needles in the haystack? A comparison of phosphotyrosine peptides enriched by immuno-affinity precipitation and metal-based affinity chromatography.

Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal-affinity based enrichment (Ti(4+)-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling. BIOLOGICAL SIGNIFICANCE: Here we evaluated enabling tools for the global analysis of phosphotyrosine phosphorylation. Phosphotyrosine phosphorylation is a key protein modification driving cellular response also involved in disease/cancer molecular pathways.