Faculty Bibliography

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors

PURPOSE: To increase knowledge of the biochemical composition of lenticular exfoliation material (XFM) by using proteomic approaches. METHODS: Anterior lens capsules from patients with and without exfoliation syndrome (XFS) were homogenized in formic acid and subjected to cyanogen bromide (CNBr) cleavage, and the pattern of chemically generated fragments was compared by SDS-PAGE after silver staining. Unique XFS bands not present in control cases were excised, digested with TPCK-trypsin, and the resultant peptides sequenced with quadrupole time-of-flight mass spectrometry (MS). In parallel experiments, CNBr-fragmented XFM was separately digested in solution with trypsin and elastase, and the resultant peptide mixture was analyzed by liquid chromatography coupled to tandem MS followed by identification through homology searches at nonredundant protein databases. Immunolocalization of the MS-identified components were performed in XFS versus control samples by using conventional immunohistochemical methods and light microscopy. RESULTS: In addition to fibrillin-1, fibronectin, vitronectin, laminin, and amyloid P-component, which are well-known extracellular matrix and basement membrane components of XFM, the proteomic approaches identified the multifunctional protein clusterin and tissue inhibitor of metalloprotease (TIMP)-3 as well as novel molecules, among them fibulin-2, desmocollin-2, the glycosaminoglycans syndecan-3, and versican, membrane metalloproteases of the ADAM family (a disintegrin and metalloprotease), and the initiation component of the classic complement activation pathway C1q. In all cases, classic immunohistochemistry confirmed their location in XFM. CONCLUSIONS: A novel solubilization strategy combined with sensitive proteomic analysis emphasizes the complexity of the XFS deposits and opens new avenues to study the molecular mechanisms involved in the pathogenesis and progression of XFS

Accurate protein identification sometimes requires careful discrimination between closely related protein isoforms that may differ by as little as a single amino acid substitution or post-translational modification. The ABRF Proteomics Research Group sent a mixture of three picomoles each of three closely related proteins to laboratories who requested it in the form of intact proteins, and participating laboratories were asked to identify the proteins and report their results. The primary goal of the ABRF-PRG04 Study was to give participating laboratories a chance to evaluate their capabilities and practices with regards to sample fractionation (1D- or 2D-PAGE, HPLC, or none), protein digestion methods (in-solution, in-gel, enzyme choice), and approaches to protein identification (instrumentation, use of software, and/or manual techniques to facilitate interpretation), as well as determination of amino acid or post-translational modifications. Of the 42 laboratories that responded, 8 (19%) correctly identified all three isoforms and N-terminal acetylation of each, 16 (38%) labs correctly identified two isoforms, 9 (21%) correctly identified two isoforms but also made at least one incorrect identification, and 9 (21%) made no correct protein identifications. All but one lab used mass spectrometry, and data submitted enabled a comparison of strategies and methods used.

We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes

We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic digestion using this method requires half the time of in-gel digestion and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional in-gel digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode, which is specifically designed for staining NC membrane-immobilized proteins and is faster and more sensitive than Ponceau-S. Our improved on-membrane digestion protocol greatly improves the study of soluble and, particularly strikingly, integral membrane proteins by mass spectrometry

Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance, and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY IPs from ephrinB1-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. On the basis of previously published work and use of PathwayAssist software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios

Comparative proteomic approaches using isotopic labeling and mass spectrometry (MS) have become increasingly popular. Conventionally, quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with 13C6 lysine and 15N4 arginine, the other set unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a ten-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG-108 cells

Identification of tyrosine phosphorylation by MS is challenging due to its low abundance in biological samples. Therefore, specific enrichment of tyrosine phosphorylated peptides prior to their analysis is highly desirable. The application of immunopurification of phosphotyrosine (pY) peptides using pY antibodies has been greatly limited by poor selectivity. In the present study, we have shown that the selectivity of pY peptide immunopurification can be dramatically improved by adding detergents to immunoprecipitation buffers. Optimum selectivity and sensitivity were achieved using an immunoprecipitation buffer containing n-octyl glucoside with a concentration above its critical micelle concentration (0.7%). The optimized method was used to identify in vivo tyrosine phosphorylation on proteins isolated from cell extract by anti-pY protein immunoprecipitation. After immunopurification, non-pY-containing peptides from protein digests were readily removed and pY peptides became the dominant peaks in MALDI quadrupole-TOF mass spectra. In addition, the signal intensities from pY-containing peptides were enhanced significantly after enrichment, allowing characterization of tyrosine phosphorylation sites with greater sensitivity