O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins.

Mycobacterium tuberculosis (Mtb) causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Cell-cell recognition between the pathogen Mtb and its host is mediated in part by glycosylated proteins. So far, glycoproteins in Mtb are understudied and for only very few glycoproteins glycosylation sites have been described, e.g., alanine and proline rich secreted protein apa, superoxide dismutase SODC, lipoprotein lpqH and MPB83/MPT83. In this study, glycosylated proteins in Mtb culture filtrate were investigated using liquid chromatography-mass spectrometry approaches and bioinformatic analyses. To validate the presence of glycoproteins, several strategies were pursued including collision induced dissociation, high energy collision dissociation and electron transfer dissociation techniques, and bioinformatics analyses involving a neutral loss search for glycosylated moieties. After extensive data curation, we report glycosylation sites for thirteen Mtb glycoproteins using a combination of mass spectrometry techniques on a dataset collected from culture filtrate proteins. This is the first glycoproteomics study identifying glycosylation sites on mycobacterial culture filtrate proteins (CFP) on a global scale. BIOLOGICAL SIGNIFICANCE: In this study, glycosylation sites in Mtb were characterized by collision-induced dissociation, electron-transfer dissociation and high energy collision dissociation techniques. The identification of glycosylation sites is important for our understanding of the physiology and pathophysiology of Mtb. Glycoproteins are often responsible for protein-protein interactions between host and pathogen and thus represent interesting targets for vaccine development. In addition, our strategy is not limited to Mtb, but could be extended to other organisms. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Evaluation and optimization of mass spectrometric settings during data-dependent acquisition mode: focus on LTQ-Orbitrap mass analyzers.

Mass-spectrometry-based proteomics has evolved as the preferred method for the analysis of complex proteomes. Undoubtedly, recent advances in mass spectrometry instrumentation have greatly enhanced proteomic analysis. A popular instrument platform in proteomics research is the LTQ-Orbitrap mass analyzer. In this tutorial, we discuss the significance of evaluating and optimizing mass spectrometric settings on the LTQ-Orbitrap during CID data-dependent acquisition (DDA) mode to improve protein and peptide identification rates. We focus on those MS and MS/MS parameters that have been systematically examined and evaluated by several researchers and are commonly used during DDA. More specifically, we discuss the effect of mass resolving power, preview mode for FTMS scan, monoisotopic precursor selection, signal threshold for triggering MS/MS events, number of microscans per MS/MS scan, number of MS/MS events, automatic gain control target value (ion population) for MS and MS/MS, maximum ion injection time for MS/MS, rapid and normal scan rate, and prediction of ion injection time. We furthermore present data from the latest generation LTQ-Orbitrap system, the Orbitrap Elite, along with recommended MS and MS/MS parameters. The Orbitrap Elite outperforms the Orbitrap Classic in terms of scan speed, sensitivity, dynamic range, and resolving power and results in higher identification rates. Several of the optimized MS parameters determined on the LTQ-Orbitrap Classic and XL were easily transferable to the Orbitrap Elite, whereas others needed to be reevaluated. Finally, the Q Exactive and HCD are briefly discussed, as well as sample preparation, LC-optimization, and bioinformatics analysis. We hope this tutorial will serve as guidance for researchers new to the field of proteomics and assist in achieving optimal results.

Designer reagents for mass spectrometry-based proteomics: clickable cross-linkers for elucidation of protein structures and interactions.

We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein-protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell-permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g., in vivo cross-linking) is illustrated by Western blot detection of Cul1 complexes including known binders, Cand1 and Skp2, in HEK 293 cells, confirming good water solubility and cell-permeability.

Click chemistry facilitates formation of reporter ions and simplified synthesis of amine-reactive multiplexed isobaric tags for protein quantification.

We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Characterization of the Mycobacterium tuberculosis proteome by liquid chromatography mass spectrometry-based proteomics techniques: a comprehensive resource for tuberculosis research.

Approximately, one-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Secreted and membrane proteins that interact with the host play important roles for the pathogenicity of the bacteria and are potential drug targets or components of vaccines. In this present study, subcellular fractionation in combination with membrane enrichment was used to comprehensively analyze the M. tuberculosis proteome. The proteome of the M. tuberculosis cell wall, membrane, cytosol, lysate, and culture filtrate was defined with a high coverage. Exceptional enrichment for membrane proteins was achieved using wheat germ agglutinin (WGA)-affinity two-phase partitioning, a technique that has to date not yet been exploited for the enrichment of mycobacterial membranes. Overall, 1051 M. tuberculosis protein groups including 183 transmembrane proteins have been identified by LC-MS/MS analysis using stringent database search criteria with a minimum of two peptides and an estimated FDR of less than 1%. With many mycobacterial antigens and lipoglycoproteins identified, the results from this study suggest that many of the newly discovered proteins could represent potential candidates mediating host-pathogen interactions. In addition, this data set provides experimental information about protein localization and thus serves as a valuable resource for M. tuberculosis proteome research.

LogViewer: a software tool to visualize quality control parameters to optimize proteomics experiments using Orbitrap and LTQ-FT mass spectrometers.

Visualization tools that allow both optimization of instrument's parameters for data acquisition and specific quality control (QC) for a given sample prior to time-consuming database searches have been scarce until recently and are currently still not freely available. To address this need, we have developed the visualization tool LogViewer, which uses diagnostic data from the RAW files of the Thermo Orbitrap and linear trap quadrupole-Fourier transform (LTQ-FT) mass spectrometers to monitor relevant metrics. To summarize and visualize the performance on our test samples, log files from RawXtract are imported and displayed. LogViewer is a visualization tool that allows a specific and fast QC for a given sample without time-consuming database searches. QC metrics displayed include: mass spectrometry (MS) ion-injection time histograms, MS ion-injection time versus retention time, MS(2) ion-injection time histograms, MS(2) ion-injection time versus retention time, dependent scan histograms, charge-state histograms, mass-to-charge ratio (M/Z) distributions, M/Z histograms, mass histograms, mass distribution, summary, repeat analyses, Raw MS, and Raw MS(2). Systematically optimizing all metrics allowed us to increase our protein identification rates from 600 proteins to routinely determine up to 1400 proteins in any 160-min analysis of a complex mixture (e.g., yeast lysate) at a false discovery rate of <1%. Visualization tools, such as LogViewer, make QC of complex liquid chromotography (LC)-MS and LC-MS/MS data and optimization of the instrument's parameters accessible to users.

Cdc48/p97 mediates UV-dependent turnover of RNA Pol II.

Cdc48/p97 is an essential ATPase whose role in targeting substrates to the ubiquitin-proteasome system (UPS) remains unclear. Existing models posit that Cdc48 acts upstream of UPS receptors. To address this hypothesis, we examined the association of ubiquitin (Ub) conjugates with 26S proteasomes. Unexpectedly, proteasomes isolated from cdc48 mutants contain high levels of Ub conjugates, and mass spectrometry identified numerous nonproteasomal proteins, including Rpb1, the largest subunit of RNA Pol II. UV-induced turnover of Rpb1 depends upon Cdc48-Ufd1-Npl4, Ubx4, and the uncharacterized adaptor Ubx5. Ubiquitinated Rpb1, proteasomes, and Cdc48 accumulate on chromatin in UV-treated wild-type cells, and the former two accumulate to higher levels in mutant cells, suggesting that degradation of Rpb1 is facilitated by Cdc48 at sites of stalled transcription. These data reveal an intimate coupling of function between proteasomes and Cdc48 that we suggest is necessary to sustain processive degradation of unstable subunits of some macromolecular protein complexes.

The steady-state repertoire of human SCF ubiquitin ligase complexes does not require ongoing Nedd8 conjugation.

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin-like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1-RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.

UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover.

p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.

A putative stimulatory role for activator turnover in gene expression.

The ubiquitin-proteasome system (UPS) promotes the destruction of target proteins by attaching to them a ubiquitin chain that is recognized by the 26S proteasome. The UPS influences most cellular processes, and its targets include transcriptional activators that are primary determinants of gene expression. Emerging evidence indicates that non-proteolytic functions of the UPS might stimulate transcriptional activity. Here we show that the proteolysis of some transcriptional activators by the UPS can stimulate their function. We focused on the role of UPS-dependent proteolysis in the function of inducible transcriptional activators in yeast, and found that inhibition of the proteasome reduced transcription of the targets of the activators Gcn4, Gal4 and Ino2/4. In addition, mutations in SCF(Cdc4), the ubiquitin ligase for Gcn4 (ref. 5), or mutations in ubiquitin that prevent degradation, also impaired the transcription of Gcn4 targets. These transcriptional defects were manifested despite the enhanced abundance of Gcn4 on cognate promoters. Proteasome inhibition also decreased the association of RNA polymerase II with Gcn4, Gal4 and Ino2/4 targets, as did mutations in SCF(Cdc4) for Gcn4 targets. Expression of a stable phospho-site mutant of Gcn4 (ref. 7) or disruption of the kinases that target Gcn4 for turnover alleviated the sensitivity of Gcn4 activity to defects in the UPS.

Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.

The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10Delta cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10Delta) and identified 54 proteins that were uniquely recovered from rpn10Delta cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely in abundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.