Optimizing electrospray interfaces using slowly diverging conical duct (ConDuct) electrodes.

We demonstrate that the efficiency of ion transmission from atmosphere to vacuum through stainless steel electrodes that contain slowly divergent conical duct (ConDuct) channels can be close to 100%. Here, we explore the properties of 2.5-cm-long electrodes with angles of divergence of 0°, 1°, 2°, 3°, 5°, 8°, 13°, and 21°, respectively.

Maximizing ion transmission from atmospheric pressure into the vacuum of mass spectrometers with a novel electrospray interface.

We have discovered that an electrode containing a conical channel with a small angular divergence can transmit into the vacuum almost 100% of an electrospray ion current produced at atmospheric pressure. Our first implementation of such a conical duct, which we term "ConDuct," uses a conductive plastic pipette tip containing an approximately 1.6° divergent channel at its entrance.

See & Catch method for studying protein complexes in yeast cells: a technique unifying fluorescence microscopy and mass spectrometry.

We have developed a method for studying proteins and protein complexes in yeast cells based on unification of fluorescence microscopy and mass spectrometry techniques. To apply the method, termed by us as "See & Catch," we first produced a variety of DNA plasmids used as PCR templates for genomic tagging of proteins with a modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes.

Quantitative encoding of the effect of a partial agonist on individual opioid receptors by multisite phosphorylation and threshold detection.

In comparison to endogenous ligands of seven-transmembrane receptors, which typically act as full agonists, many drugs act as partial agonists. Partial agonism is best described as a "macroscopic" property that is manifest at the level of physiological systems or cell populations; however, whether partial agonists also encode discrete regulatory information at the "microscopic" level of individual receptors is not known. Here, we addressed this question by focusing on morphine, a partial agonist drug for μ-type opioid peptide receptors (MORs), and by combining quantitative mass spectrometry with cell biological analysis to investigate the reduced efficacy of morphine, compared to that of a peptide full agonist, in promoting receptor endocytosis.

High-Capacity Ion Trap Coupled to a Time-of-Flight Mass Spectrometer for Comprehensive Linked Scans with no Scanning Losses.

A high-capacity ion trap coupled to a time-of-flight (TOF) mass spectrometer has been developed to carry out comprehensive linked scan analysis of all stored ions in the ion trap. The approach involves a novel tapered geometry high-capacity ion trap that can store more than 10(6) ions (range 800-4000 m/z) without degrading its performance. Ions are stored and scanned out from the high-capacity ion trap as a function of m/z, collisionally fragmented and analyzed by TOF.

Unifying fluorescence microscopy and mass spectrometry for studying protein complexes in cells.

We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes.

A role for protein phosphorylation in cytochrome P450 3A4 ubiquitin-dependent proteasomal degradation.

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation.

Ku80 removal from DNA through double strand break-induced ubiquitylation.

The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown.

Positive feedback sharpens the anaphase switch.

At the onset of anaphase, sister-chromatid cohesion is dissolved abruptly and irreversibly, ensuring that all chromosome pairs disjoin almost simultaneously. The regulatory mechanisms that generate this switch-like behaviour are unclear. Anaphase is initiated when a ubiquitin ligase, the anaphase-promoting complex (APC), triggers the destruction of securin, thereby allowing separase, a protease, to disrupt sister-chromatid cohesion.

A novel high-capacity ion trap-quadrupole tandem mass spectrometer.

We describe a prototype tandem mass spectrometer that is designed to increase the efficiency of linked-scan analyses by >100-fold over conventional linked-scan instruments. The key element of the mass spectrometer is a novel high ion capacity ion trap, combined in tandem configuration with a quadrupole collision cell and a quadrupole mass analyzer (i.e.

Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly.

Epigenetic gene silencing in eukaryotes is regulated in part by lysine methylation of the core histone proteins. While histone lysine methylation is known to control gene expression through the recruitment of modification-specific effector proteins, it remains unknown whether nonhistone chromatin proteins are targets for similar modification-recognition systems. Here we show that the histone H3 methyltransferase G9a contains a conserved methylation motif with marked sequence similarity to H3 itself.

Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes.

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components.

Cell-cycle-dependent phosphorylation of the nuclear pore Nup107-160 subcomplex.

The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes.

Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA.

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR.

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response.

Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry.

We describe a strategy, which we term hypothesis-driven multiple-stage mass spectrometry (HMS-MS), for the sensitive detection and identification of phosphopeptides derived from enzymatic digests of phosphoproteins. In this strategy, we postulate that any or all of the potential sites of phosphorylation in a given protein may be phosphorylated. Using this assumption, we calculate the m/z values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the enzyme employed for proteolysis.

POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex.

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1).

"De novo" peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: a preliminary study.

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal.

Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement.

Polycomb group protein Ezh2 is an essential epigenetic regulator of embryonic development in mice, but its role in the adult organism is unknown. High expression of Ezh2 in developing murine lymphocytes suggests Ezh2 involvement in lymphopoiesis. Using Cre-mediated conditional mutagenesis, we demonstrated a critical role for Ezh2 in early B cell development and rearrangement of the immunoglobulin heavy chain gene (Igh).

Proteomic analysis of the mammalian nuclear pore complex.

As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components.

On the nature of the chemical noise in MALDI mass spectra.

The so-called "chemical noise background" imposes a major limit on the practical sensitivity of MALDI mass spectrometry. Typically, as the amount of material of interest subjected to MALDI analysis is reduced, the signal decreases to the point where it can no longer be differentiated from the chemical noise. Using a newly designed MALDI-ion trap mass spectrometer, we describe experiments intended to throw light on the nature of the chemical noise background and to reduce its effects.

SYT associates with human SNF/SWI complexes and the C-terminal region of its fusion partner SSX1 targets histones.

A global transcriptional co-activator, the SNF/SWI complex, has been characterized as a chromatin remodeling factor that enhances accessibility of the transcriptional machinery to DNA within a repressive chromatin structure. On the other hand, mutations in some human SNF/SWI complex components have been linked to tumor formation. We show here that SYT, a partner protein generating the synovial sarcoma fusion protein SYT-SSX, associates with native human SNF/SWI complexes.