Novel integrated computational AMP discovery approaches highlight diversity in the helminth AMP repertoire.

Antimicrobial Peptides (AMPs) are immune effectors that are key components of the invertebrate innate immune system providing protection against pathogenic microbes. Parasitic helminths (phylum Nematoda and phylum Platyhelminthes) share complex interactions with their hosts and closely associated microbiota that are likely regulated by a diverse portfolio of antimicrobial immune effectors including AMPs. Knowledge of helminth AMPs has largely been derived from nematodes, whereas the flatworm AMP repertoire has not been described. This study highlights limitations in the homology-based approaches, used to identify putative nematode AMPs, for the characterisation of flatworm AMPs, and reveals that innovative algorithmic AMP prediction approaches provide an alternative strategy for novel helminth AMP discovery. The data presented here: (i) reveal that flatworms do not encode traditional lophotrochozoan AMP groups (Big Defensin, CSαß peptides and Myticalin); (ii) describe a unique integrated computational pipeline for the discovery of novel helminth AMPs; (iii) reveal >16,000 putative AMP-like peptides across 127 helminth species; (iv) highlight that cysteine-rich peptides dominate helminth AMP-like peptide profiles; (v) uncover eight novel helminth AMP-like peptides with diverse antibacterial activities, and (vi) demonstrate the detection of AMP-like peptides from Ascaris suum biofluid. These data represent a significant advance in our understanding of the putative helminth AMP repertoire and underscore a potential untapped source of antimicrobial diversity which may provide opportunities for the discovery of novel antimicrobials. Further, unravelling the role of endogenous worm-derived antimicrobials and their potential to influence host-worm-microbiome interactions may be exploited for the development of unique helminth control approaches.

Generation of a mouse SWATH-MS spectral library to quantify 10148 proteins involved in cell reprogramming.

Murine models are amongst the most widely used systems to study biology and pathology. Targeted quantitative proteomic analysis is a relatively new tool to interrogate such systems. Recently the need for relative quantification on hundreds to thousands of samples has driven the development of Data Independent Acquisition methods. One such technique is SWATH-MS, which in the main requires prior acquisition of mass spectra to generate an assay reference library. In stem cell research, it has been shown pluripotency can be induced starting with a fibroblast population. In so doing major changes in expressed proteins is inevitable. Here we have created a reference library to underpin such studies. This is inclusive of an extensively documented script to enable replication of library generation from the raw data. The documented script facilitates reuse of data and adaptation of the library to novel applications. The resulting library provides deep coverage of the mouse proteome. The library covers 29519 proteins (53% of the proteome) of which 7435 (13%) are supported by a proteotypic peptide.

Mitochondrial fusion is required for spermatogonial differentiation and meiosis.

Differentiating cells tailor their metabolism to fulfill their specialized functions. We examined whether mitochondrial fusion is important for metabolic tailoring during spermatogenesis. Acutely after depletion of mitofusins Mfn1 and Mfn2, spermatogenesis arrests due to failure to accomplish a metabolic shift during meiosis. This metabolic shift includes increased mitochondrial content, mitochondrial elongation, and upregulation of oxidative phosphorylation (OXPHOS). With long-term mitofusin loss, all differentiating germ cell types are depleted, but proliferation of stem-like undifferentiated spermatogonia remains unaffected. Thus, compared with undifferentiated spermatogonia, differentiating spermatogonia and meiotic spermatocytes have cell physiologies that require high levels of mitochondrial fusion. Proteomics in fibroblasts reveals that mitofusin-null cells downregulate respiratory chain complexes and mitochondrial ribosomal subunits. Similarly, mitofusin depletion in immortalized spermatocytes or germ cells in vivo results in reduced OXPHOS subunits and activity. We reveal that by promoting OXPHOS, mitofusins enable spermatogonial differentiation and a metabolic shift during meiosis.

Diagnosis of epithelial ovarian cancer using a combined protein biomarker panel.

BACKGROUND: An early detection tool for EOC was constructed from analysis of biomarker expression data from serum collected during the UKCTOCS. METHODS: This study included 49 EOC cases (19 Type I and 30 Type II) and 31 controls, representing 482 serial samples spanning seven years pre-diagnosis. A logit model was trained by analysis of dysregulation of expression data of four putative biomarkers, (CA125, phosphatidylcholine-sterol acyltransferase, vitamin K-dependent protein Z and C-reactive protein); by scoring the specificity associated with dysregulation from the baseline expression for each individual. RESULTS: The model is discriminatory, passes k-fold and leave-one-out cross-validations and was further validated in a Type I EOC set. Samples were analysed as a simulated annual screening programme, the algorithm diagnosed cases with >30% PPV 1-2 years pre-diagnosis. For Type II cases (~80% were HGS) the algorithm classified 64% at 1 year and 28% at 2 years tDx as severe. CONCLUSIONS: The panel has the potential to diagnose EOC one-two years earlier than current diagnosis. This analysis provides a tangible worked example demonstrating the potential for development as a screening tool and scrutiny of its properties. Limits on interpretation imposed by the number of samples available are discussed.

A combined biomarker panel shows improved sensitivity for the early detection of ovarian cancer allowing the identification of the most aggressive type II tumours.

BACKGROUND: There is an urgent need for biomarkers for the early detection of ovarian cancer (OC). The purpose of this study was to assess whether changes in serum levels of lecithin-cholesterol acyltransferase (LCAT), sex hormone-binding globulin (SHBG), glucose-regulated protein, 78 kDa (GRP78), calprotectin and insulin-like growth factor-binding protein 2 (IGFBP2) are observed before clinical presentation and to assess the performance of these markers alone and in combination with CA125 for early detection. METHODS: This nested case-control study used samples from the United Kingdom Collaborative Trial of Ovarian Cancer Screening trial. The sample set consisted of 482 serum samples from 49 OC subjects and 31 controls, with serial samples spanning up to 7 years pre-diagnosis. The set was divided into the following: (I) a discovery set, which included all women with only two samples from each woman, the first at<14 months and the second at >32 months to diagnosis; and (ii) a corroboration set, which included all the serial samples from the same women spanning the 7-year period. Lecithin-cholesterol acyltransferase, SHBG, GRP78, calprotectin and IGFBP2 were measured using ELISA. The performance of the markers to detect cancers pre-diagnosis was assessed. RESULTS: A combined threshold model IGFBP2 >78.5 ng ml-1 : LCAT <8.831 µg ml-1 : CA125 >35 U ml-1 outperformed CA125 alone for the earlier detection of OC. The threshold model was able to identify the most aggressive Type II cancers. In addition, it increased the lead time by 5-6 months and identified 26% of Type I subjects and 13% of Type II subjects that were not identified by CA125 alone. CONCLUSIONS: Combined biomarker panels (IGFBP2, LCAT and CA125) outperformed CA125 up to 3 years pre-diagnosis, identifying cancers missed by CA125, providing increased diagnostic lead times for Type I and Type II OC. The model identified more aggressive Type II cancers, with women crossing the threshold dying earlier, indicating that these markers can improve on the sensitivity of CA125 alone for the early detection of OC.

Novel risk models for early detection and screening of ovarian cancer.

PURPOSE: Ovarian cancer (OC) is the most lethal gynaecological cancer. Early detection is required to improve patient survival. Risk estimation models were constructed for Type I (Model I) and Type II (Model II) OC from analysis of Protein Z, Fibronectin, C-reactive protein and CA125 levels in prospectively collected samples from the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). RESULTS: Model I identifies cancers earlier than CA125 alone, with a potential lead time of 3-4 years. Model II detects a number of high grade serous cancers at an earlier stage (Stage I/II) than CA125 alone, with a potential lead time of 2-3 years and assigns high risk to patients that the ROCA Algorithm classified as normal. MATERIALS AND METHODS: This nested case control study included 418 individual serum samples serially collected from 49 OC cases and 31 controls up to six years pre-diagnosis. Discriminatory logit models were built combining the ELISA results for candidate proteins with CA125 levels. CONCLUSIONS: These models have encouraging sensitivities for detecting pre-clinical ovarian cancer, demonstrating improved sensitivity compared to CA125 alone. In addition we demonstrate how the models improve on ROCA for some cases and outline their potential future use as clinical tools.

Protein Z: A putative novel biomarker for early detection of ovarian cancer.

Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded-evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down-regulated up to 2 years pre-diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up-regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA-125 and CA-125 combined with Protein Z showed a statistically significant (p = 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA-125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes.

Cell-specific proteomic analysis in Caenorhabditis elegans.

Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.

Semiquantitative analysis of clinical heat stress in Clostridium difficile strain 630 using a GeLC/MS workflow with emPAI quantitation.

Clostridium difficile is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 C. difficile proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.

Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni.

Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication. BIOLOGICAL SIGNIFICANCE: This work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.

F-box protein FBXL16 binds PP2A-B55α and regulates differentiation of embryonic stem cells along the FLK1+ lineage.

The programmed formation of specific tissues from embryonic stem cells is a major goal of regenerative medicine. To identify points of intervention in cardiac tissue formation, we performed an siRNA screen in murine embryonic stem cells to identify ubiquitin system genes that repress cardiovascular tissue formation. Our screen uncovered an F-box protein, Fbxl16, as a repressor of one of the earliest steps in the cardiogenic lineage: FLK1+ progenitor formation. Whereas F-box proteins typically form SCF ubiquitin ligases, shotgun mass spectrometry revealed that FBXL16 instead binds protein phosphatase 2A (PP2A) containing a B55 specificity subunit (PP2A(B55)). Phosphoproteomic analyses indicate that FBXL16 negatively regulates phosphorylation of the established PP2A(B55) substrate, vimentin. We suggest that FBXL16 negatively regulates the activity of B55α-PP2A to modulate the genesis of FLK1+ progenitor cells.

Identification of secreted bacterial proteins by noncanonical amino acid tagging.

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

Perturbations to the ubiquitin conjugate proteome in yeast δubx mutants identify Ubx2 as a regulator of membrane lipid composition.

Yeast Cdc48 (p97/VCP in human cells) is a hexameric AAA ATPase that is thought to use ATP hydrolysis to power the segregation of ubiquitin-conjugated proteins from tightly bound partners. Current models posit that Cdc48 is linked to its substrates through adaptor proteins, including a family of seven proteins (13 in human) that contain a Cdc48-binding UBX domain. However, few substrates for specific UBX proteins are known, and hence the generality of this hypothesis remains untested. Here, we use mass spectrometry to identify ubiquitin conjugates that accumulate in cdc48 and ubx mutants. Different ubx mutants exhibit unique patterns of conjugate accumulation that point to functional specialization of individual Ubx proteins. To validate our findings, we examined in detail the endoplasmic reticulum-bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. Mutant ubx2Δ cells are deficient in both cleaving the ubiquitinated 120 kDa precursor of Spt23 to form active p90 and in localizing p90 to the nucleus, resulting in reduced expression of the target gene OLE1, which encodes fatty acid desaturase. Our findings provide a resource for future investigations on Cdc48, illustrate the utility of proteomics to identify ligands for specific ubiquitin receptor pathways, and uncover Ubx2 as a key player in the regulation of membrane lipid biosynthesis.

Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins.

The modular SCF (Skp1, cullin, and F box) ubiquitin ligases feature a large family of F box protein substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCF(Fbxw7) is extraordinarily stable, but, in the Nedd8-deconjugated state, the cullin-binding protein Cand1 augments its dissociation by one-million-fold. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1-Rbx1 equilibrates with multiple F box protein-Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.

Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment.

The adenocarcinoma cell line HeLa serves as a model system for cancer research in general and cervical cancer in particular. In this study, hydrazide enrichment in combination with state-of-the art nanoLC-MS/MS analysis was used to profile N-linked glycosites in HeLa cells. N-Linked glycoproteins were selectively enriched in HeLa cells by the hydrazide capture method, which isolates all glycoproteins independent of their glycans. Nonglycosylated proteins were removed by extensive washing. N-Linked glycoproteins were identified with the specific NXT/S motif and deamidated asparagine (N). Deglycosylation was carried out in both H(2)(16)O and H(2)(18)O to confirm the deamidation. NanoLC-MS/MS analysis indicated that the method selectively enriched at least 100 fold N-linked glycosites in HeLa cells. When both the membrane and cytosolic fractions were used, a total of 268 unique N-glycosylation sites were identified corresponding to 106 glycoproteins. Bioinformatic analysis revealed that most of the glycoproteins identified are known to have an impact on cancer and have been proposed as biomarkers.

Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking.

UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53.

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.

Novel proteomic tools reveal essential roles of SRP and importance of proper membrane protein biogenesis.

The signal recognition particle (SRP), which mediates cotranslational protein targeting to cellular membranes, is universally conserved and essential for bacterial and mammalian cells. However, the current understanding of the role of SRP in cell physiology and pathology is still poor, and the reasons behind its essential role in cell survival remain unclear. Here, we systematically analyzed the consequences of SRP loss in E. coli using time-resolved quantitative proteomic analyses. A series of snapshots of the steady-state and newly synthesized proteome unveiled three stages of cellular responses to SRP depletion, and demonstrated essential roles of SRP in metabolism, membrane potential, and protein and energy homeostasis in both the membrane and cytoplasm. We also identified a group of periplasmic proteins, including key molecular chaperones, whose localization was impaired by the loss of SRP; this and additional results showed that SRP is crucial for protein homeostasis in the bacterial envelope. These results reveal the extensive roles that SRP plays in bacterial physiology, emphasize the importance of proper membrane protein biogenesis, and demonstrate the ability of time-resolved quantitative proteomic analysis to provide new biological insights.

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.