Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin ␤1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using

Ig class switch recombination (CSR) and somatic hypermutation serve to diversify antibody responses and are orchestrated by the activity of activation-induced cytidine deaminase and many proteins involved in DNA repair and genome surveillance. Msh5, a gene encoded in the central MHC class III region, and its obligate heterodimerization partner Msh4 have a critical role in regulating meiotic homologous recombination and have not been implicated in CSR. Here, we show that MRL/lpr mice carrying a congenic H-2 b/b MHC interval exhibit several abnormalities regarding CSR, including a profound deficiency of IgG3 in most mice and long microhomologies at Ig switch (S) joints. We found that Msh5 is expressed at low levels on the H-2 b haplotype and, importantly, a similar long S joint microhomology phenotype was observed in both Msh5 and Msh4-null mice. We also present evidence that genetic variation in MSH5 is associated with IgA deficiency and common variable immune deficiency (CVID) in humans. One of the human MSH5 alleles identified contains two nonsynonymous polymorphisms, and the variant protein encoded by this allele shows impaired binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA deficiency patients carrying disease-associated MSH5 alleles show increased donor/acceptor microhomology, involving pentameric DNA repeat sequences and lower mutation rates than controls. Our findings suggest that Msh4/5 heterodimers contribute to CSR and support a model whereby Msh4/5 promotes the resolution of DNA breaks with low or no terminal microhomology by a classical nonhomologous end-joining mechanism while possibly suppressing an alternative microhomology-mediated pathway.immunoglobulin subclass deficiency ͉ mismatch repair ͉ Msh4 A fter appropriate stimulation, B cells undergo class switch recombination (CSR), whereby the functionally rearranged V(D)J DNA segment is recombined with a downstream Ig constant region segment. The biochemistry of CSR is complex and involves the B cell-specific gene activation-induced cytidine deaminase, which initiates both CSR and somatic hypermutation (1). CSR also requires many ubiquitously expressed genes important for detecting DNA mismatches and breaks and regulating DNA repair (2). CSR occurs at specific DNA segments called switch (S) regions, which lie upstream of each constant region and contain hotspots for activation-induced cytidine deaminase-mediated cytosine deamination. The ligation of the S region with the downstream S regions is carried out by protein factors that comprise the nonhomologous end joining machinery for DNA repair (1, 2).Mismatch repair proteins play a critical role in safeguarding genetic stability. The key proteins for initiation of eukaryotic mismatch repair are homologues of bacterial MutS and MutL. In mammals, there are five MutS (Msh2, Msh3, Msh4, Msh5, and Msh6) and four MutL (Mlh1, Mlh3, Pms1, and Pms2) homologues. Each Mut homologue acts at the DNA repair or recombination site by forming heterodimers; Msh2-Msh6 (MutS␣), Msh2-Msh3 (MutS␤), M...

Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes.

Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" ( 12 C-labeled) or "heavy" ( 13 C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.Phosphorylation and dephosphorylation are two major regulatory mechanisms controlling the activity of proteins. A number of signaling pathways rely on the activity of tyrosine kinases for transmitting signals from the cell surface to the nucleus (1). For instance, ligand binding to receptor proteintyrosine kinases activates their kinase activity and promotes their autophosphorylation. This creates docking sites for substrates that either possess intrinsic enzymatic activity or serve as adapter molecules that mediate the interaction between signaling molecules and the receptors (2). Activation of downstream tyrosine kinases enables the propagation of these signals by modifying the activity of downstream intermediates and inducing the assembly or disassembly of signaling multiprotein complexes.Identification of substrates of tyrosine kinases is a major step in the analysis of signaling pathways. Enrichment of tyrosine-phosphorylated proteins with antiphosphotyrosine antibodies is a necessary step given the low stoichiometry of tyrosine phosphorylation and the complexity of the samples such as cell lysates. In traditional approaches, immunoprecipitated protein samples derived from untreated or ligand-stimulated cells are separated in parallel by gel electrophoresis and visualized by gel staining. The differentially stained protein bands are subsequently analyzed by mass spectrometry (3-7). Nonspecific binding and binding of basally tyrosine-phosphorylated proteins to antib...