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. The ion transmission efficiency was observed to jump from 10-20% for the 0° (straight) channels to 90-95% for channels with an angle of divergence as small as 1°. Furthermore, the 2-3° ConDuct electrodes produced extraordinarily low divergence ion beams that propagated in a laser-like fashion over long distances in vacuum. To take advantage of these newly discovered properties, we constructed a novel atmosphere-to-vacuum ion interface utilizing a 2° ConDuct as an inlet electrode and compared its ion transmission efficiency with that of the interface used in the commercial (Thermo Fisher Scientific, San Jose, CA, USA) Velos Orbitrap and Q Exactive mass spectrometers. We observed that the ConDuct interface transmitted up to 17 times more ions than the commercial reference interface and also yielded improved signal-to-noise mass spectra of peptides. We infer from these results that the performance of many current atmosphere-to-vacuum interfaces utilizing metal capillaries can be substantially improved by replacing them with 1° or 2° metal ConDuct electrodes, which should preserve the convenience of supplying ion desolvation energy by heating the electrode while greatly increasing the efficiency of ion transmission into the mass spectrometer.

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. We observed that the beam formed by the ConDuct electrode has a very low divergence (less than 1°) and persists for long distances in vacuum. Intrigued by these properties, we incorporated this electrode into a novel atmosphere-to-vacuum ion transmission interface, and devised a technique for evaluating its performance relative to the commercial reference interfaces that contain heated metal capillaries. We determined that our new interface transmits at least 400 times more ions than the commercial Thermo LCQ DECA XP atmosphere-to-vacuum interface and 2 to 3 times more than the commercial interface in the Thermo Velos Orbitrap and the Q Exactive mass spectrometers. We conclude that it might be possible to optimize the properties of the transmitted ions further by manufacturing ConDuct inlet electrodes from metal rather than conductive plastic and by determining the optimum angle of channel divergence and channel length.

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. Among those modular tags, we found several combinations which were optimal for determining protein subcellular localization and for purifying the tagged proteins and protein complexes for detailed analysis by mass spectrometry. Combining fluorescence microscopy and mass spectrometry into a single method provides a unique possibility to obtain a unified view of the processes regulating dynamic properties of the proteins and protein complexes in living cells.

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. We showed that these chemically distinct ligands produced a complex and qualitatively similar mixture of phosphorylated opioid receptor forms in intact cells. Quantitatively, however, the different agonists promoted disproportionate multisite phosphorylation of a specific serine and threonine motif, and we found that modification at more than one residue was essential for the efficient recruitment of the adaptor protein ß-arrestin that mediated subsequent endocytosis of MORs. Thus, quantitative encoding of agonist-selective endocytosis at the level of individual opioid receptors was based on the conserved biochemical principles of multisite phosphorylation and threshold detection.

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. Accurate mass analysis is achieved on both the precursor and fragment ions of all species ejected from the ion trap. We demonstrate the approach for comprehensive linked-scan identification of phosphopeptides in mixtures with their corresponding unphosphorylated peptides.

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. After this step we tested the constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosis in Saccharomyces cerevisiae, with a variety of fluorescent and affinity probes. Among the modular tags produced we found several combinations that were optimal for determining subcellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and dephosphorylation. Our results indicate that this approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.

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. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr(264) and Ser(420). We now document that liver cytosolic kinases additionally target Ser(478) as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Delta strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser(478), Thr(264), and Ser(420) residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and 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. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1-Cul1-F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.

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. Here we demonstrate that the cyclin-dependent kinase 1 (Cdk1)-dependent phosphorylation of securin near its destruction-box motif inhibits securin ubiquitination by the APC. The phosphatase Cdc14 reverses securin phosphorylation, thereby increasing the rate of securin ubiquitination. Because separase is known to activate Cdc14 (refs 5 and 6), our results support the existence of a positive feedback loop that increases the abruptness of anaphase. Consistent with this model, we show that mutations that disrupt securin phosphoregulation decrease the synchrony of chromosome segregation. Our results also suggest that coupling securin degradation with changes in Cdk1 and Cdc14 activities helps coordinate the initiation of sister-chromatid separation with changes in spindle dynamics.

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. As with methylation of H3 lysine 9, autocatalytic G9a methylation is necessary and sufficient to mediate in vivo interaction with the epigenetic regulator heterochromatin protein 1 (HP1), and this methyl-dependent interaction can be reversed by adjacent G9a phosphorylation. NMR analysis indicates that the HP1 chromodomain recognizes methyl-G9a through a binding mode similar to that used in recognition of methyl-H3K9, demonstrating that the chromodomain functions as a generalized methyl-lysine binding module. These data reveal histone-like modification cassettes - or "histone mimics" - as a distinct class of nonhistone methylation targets and directly extend the principles of the histone code to the regulation of nonhistone proteins.

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. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.

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. In this study, we examined the cell-cycle-dependent phosphorylation of the Nup107-160 subcomplex, a core building block of the NPC. Using in vivo (32)P labeling in HeLa cells, we found that Nup107, Nup96, and Nup133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS(2), and MS(3)), establishing that Nup160, Nup133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS(2) analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G(1) cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107-160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107-160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins.

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. a TrapqQ configuration). This ion trap can store >10(6) ions without significant degradation of its performance. The current mass resolution of the trap is 100-450 full width at half maximum for ions in the range 800-4000 m/z, yielding a 10-20 m/z selection window for ions ejected at any given time into the collision cell. The sensitivity of the mass spectrometer for detecting peptides is in the low femtomole range. We can envisage relatively straightforward modifications to the instrument that should improve both its resolution and sensitivity. We tested the tandem mass spectrometer for collecting precursor ion spectra of all the ions stored in the trap and demonstrated that we can selectively detect a phosphopeptide in a mixture of non-phosphorylated peptides. Based on this prototype instrument, we plan to construct a fully functional model of the mass spectrometer for detecting modification sites on proteins and profiling their abundances with high speed and sensitivity.

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. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

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. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.

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. We test ions at these m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry (MS(2)) in a vacuum MALDI ion trap mass spectrometer, where the neutral loss of the elements of H(3)PO(4) (98 Da) provides a sensitive assay for the presence of phosphopeptides. Subsequent MS(3) analysis of the (M + H - 98)(+) peaks allows us to confirm or reject the hypotheses that the putative phosphopeptides are present in the sample. HMS-MS was successfully applied to the detection and identification of phosphopeptides from substrates of the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6), basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro. The method was also used to find in vitro phosphopeptides from a domain of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr residues without assuming a consensus motif. Our results demonstrate that HMS-MS is a sensitive, highly specific tool for systematically surveying proteins for Ser/Thr phosphorylation, and represents a significant step toward our goal of comprehensive phosphorylation mapping.

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). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.

"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. Chem. 2001, 73, 5066-5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-to-noise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process.

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). We also revealed Ezh2 as a key regulator of histone H3 methylation in early B cell progenitors. Our data suggest Ezh2-dependent histone H3 methylation as a novel regulatory mechanism controlling Igh rearrangement during early murine B cell development.

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. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.