Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature.

Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization-mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a two-column setup was evaluated for improvements in chromatography. Using commercially available standards, we demonstrate that a low column temperature (0 °C) during sample loading enhances the peak shape of several bovine serum albumin hydrophilic peptides. For digested HeLa lysates, approximately 15% more peptide identifications were obtained by increasing the precolumn temperature to 50 °C after the 500 ng sample was loaded at a low temperature. This method also identified additional early eluting peptides with grand average of hydropathicity values less than -2. We also investigated the effect of cooler column temperatures on peptides with post-translational modifications. It was possible to minimize the coelution of an isoaspartylated peptide and its unmodified version when the analytical column temperature was decreased to 5 °C. Aside from demonstrating the utility of lower temperatures for improved chromatography, its application at specific locations and time points is critical for peptide detection and separation.

Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export.

Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3'-poly(A)-tail of messenger RNAs. Here, we characterize Pab1 as an export cargo of the protein export factor Xpo1/Crm1. Pab1 is a major Xpo1/Crm1-interacting protein in yeast extracts and binds directly to Xpo1/Crm1 in a RanGTP-dependent manner. Pab1 shuttles rapidly between the nucleus and the cytoplasm and partially accumulates in the nucleus when the function of Xpo1/Crm1 is inhibited. However, Pab1 can also be exported by an alternative pathway, which is dependent on the MEX67-mRNA export pathway. Import of Pab1 is mediated by the import receptor Kap108/Sxm1 through a nuclear localization signal in its fourth RNA-binding domain. Interestingly, inhibition of Pab1's nuclear import causes a kinetic delay in the export of mRNA. Furthermore, the inviability of a pab1 deletion strain is suppressed by a mutation in the 5'-3' exoribonuclease RRP6, a component of the nuclear exosome. Therefore, nuclear Pab1 may be required for efficient mRNA export and may function in the quality control of mRNA in the nucleus.

Proteomic mapping of brain plasma membrane proteins.

Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.

Experimental Peptide Identification Repository (EPIR): an integrated peptide-centric platform for validation and mining of tandem mass spectrometry data.

LC MS/MS has become an established technology in proteomic studies, and with the maturation of the technology the bottleneck has shifted from data generation to data validation and mining. To address this bottleneck we developed Experimental Peptide Identification Repository (EPIR), which is an integrated software platform for storage, validation, and mining of LC MS/MS-derived peptide evidence. EPIR is a cumulative data repository where precursor ions are linked to peptide assignments and protein associations returned by a search engine (e.g. Mascot, Sequest, or PepSea). Any number of datasets can be parsed into EPIR and subsequently validated and mined using a set of software modules that overlay the database. These include a peptide validation module, a protein grouping module, a generic module for extracting quantitative data, a comparative module, and additional modules for extracting statistical information. In the present study, the utility of EPIR and associated software tools is demonstrated on LC MS/MS data derived from a set of model proteins and complex protein mixtures derived from MCF-7 breast cancer cells. Emphasis is placed on the key strengths of EPIR, including the ability to validate and mine multiple combined datasets, and presentation of protein-level evidence in concise, nonredundant protein groups that are based on shared peptide evidence.

Screening for N-glycosylated proteins by liquid chromatography mass spectrometry.

In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.

Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability.

DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilization upon genotoxic stress. We describe a distinct, markedly stable form of Cdc25A, which plays a previously unrecognized role in mitosis. Mitotic stabilization of Cdc25A reflects its phosphorylation on Ser17 and Ser115 by cyclin B-Cdk1, modifications required to uncouple Cdc25A from its ubiquitin-proteasome-mediated turnover. Cdc25A binds and activates cyclin B-Cdk1, accelerates cell division when overexpressed, and its downregulation by RNA interference (RNAi) delays mitotic entry. DNA damage-induced G(2) arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G(2) checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles for Cdc25A in multiple cell cycle transitions and checkpoints.

Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases.

We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.

Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development.

The LIM-domain-binding protein Ldb1 is a key factor in the assembly of transcriptional complexes involving LIM-homeodomain proteins and other transcription factors that regulate animal development. We identified Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) as components of Ldb1-associated nuclear complexes in HeLa cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. This association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from Drosophila to humans. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila Ssdp gene is unique. In Xenopus, Ssdp encoded by Drosophila Ssdp or mouse Ssdp1 mRNA enhances axis induction by Ldb1 in conjunction with the LIM-homeobox gene Xlim1. Furthermore, we were able to demonstrate an interaction between Ssdp and Chip (the fly homolog of Ldb1) in Drosophila wing development. These findings indicate functional conservation of Ssdp as a cofactor of Ldb1 during invertebrate and vertebrate development.

Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck.

Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. Here we report a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 (p53-inducible messenger RNA with a relative molecular mass (M(r)) of 140,000), Nap125 (NCK-associated protein with an M(r) of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. We therefore propose that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation.

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry.

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.