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Helicobacter pylori colonizes the human stomach and is associated with an increased risk of gastric cancer and peptic ulcer disease. Analysis of H. pylori protein secretion is complicated by the occurrence of bacterial autolysis. In this study, we analyzed the exoproteome of H. pylori at multiple phases of bacterial growth and identified 74 proteins that are selectively released into the extracellular space. These include proteins known to cause alterations in host cells, antigenic proteins, and additional proteins that have not yet been studied in any detail. The composition of the H. pylori exoproteome is dependent on the phase of bacterial growth. For example, the proportional abundance of the vacuolating toxin VacA in culture supernatant is higher during late growth phases than early growth phases, whereas the proportional abundance of many other proteins is higher during early growth phases. We detected marked variation in the subcellular localization of putative secreted proteins within soluble and membrane fractions derived from intact bacteria. By providing a comprehensive view of the H. pylori exoproteome, these results provide new insights into the array of secreted H. pylori proteins that may cause alterations in the gastric environment. [ABSTRACT FROM AUTHOR]

We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes, PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during impart, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1 p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner. [ABSTRACT FROM AUTHOR]

Cancer research depends on the use of human cell lines for both the in vitro (culture) and in vivo (xenograft) analysis of tumor progression and treatment. However, the extent to which cultured preparations of human cancer lines display similar properties in vivo, where important host factors may influence tumor biology, remains unclear. Here, we address this question by conducting a functional proteomic analysis of the human breast cancer line MDA-MB-231 grown in culture and as orthotopic xenograft tumors in the mammary fad pad of immunodeficient mice. Using a suite of activity-based chemical probes, we identified carcinoma (human) enzyme activities that were expressed selectively in culture or in xenograft tumors. Likewise, distinct groups of stromal (mouse) enzyme activities were found that either infiltrated or were excluded from xenograft tumors, indicating a contribution by specific host components to breast cancer development. MDA-MB-231 cells isolated from tumors exhibited profound differences in their enzyme activity profiles compared with the parent cell line, including the dramatic posttranscriptional up-regulation of the serine proteases urokinase plasminogen activator and tissue plasminogen activator and down-regulation of the glycolytic enzyme phosphofructokinase. These altered enzyme activity profiles correlated with significantly greater tumor growth rates and metastases for xenograft-derived MDA-MB-231 cells upon reintroduction into mice. Collectively, these data indicate that the in vivo environment of the mouse mammary fat pad cultivates the growth of human breast cancer cells with elevated tumorigenic properties and highlight the value of activity-based protein profiling for identifying proteomic signatures that depict such changes in cancer cell biology. [ABSTRACT FROM AUTHOR]

Background: Mucinous adenocarcinoma of the salivary gland (MAC) is a lethal cancer with unknown molecular etiology and a high propensity to lymph node metastasis. Mostly due to its orphan status, MAC remains one of the least explored cancers that lacks cell lines and mouse models that could help translational and pre-clinical studies. Surgery with or without radiation remains the only treatment modality but poor overall survival (10-year, 44%) underscores the urgent need for mechanism-based therapies.Methods: We developed the first patient-derived xenograft (PDX) model for pre-clinical MAC studies and a cell line that produces aggressively growing tumors after subcutaneous injection into nude mice. We performed cytogenetic, exome, and proteomic profiling of MAC to identify driving mutations, therapeutic targets, and pathways involved in aggressive cancers based on TCGA database mining and GEO analysis.Results: We identified in MAC KRAS (G13D) and TP53 (R213X) mutations that have been previously reported as drivers in a variety of highly aggressive cancers. Somatic mutations were also found in KDM6A, KMT2D, and other genes frequently mutated in colorectal and other cancers: FAT1, NBEA, RELN, RLP1B, and ZFHX3. Proteomic analysis of MAC implied epigenetic up-regulation of a genetic program involved in proliferation and cancer stem cell maintenance.Conclusion: Genomic and proteomic analyses provided the first insight into potential molecular drivers of MAC metastases pointing at common mechanisms of CSC propagation in aggressive cancers. The in vitro/in vivo models that we created should aid in the development and validation of new treatment strategies against MAC. [ABSTRACT FROM AUTHOR]

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate: ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol: fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation. [ABSTRACT FROM AUTHOR]

Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers. [Display omitted] • PAX3-FOXO1 degradation leads to cell death and myogenic differentiation • PAX3-FOXO1 degradation causes loss of transcription of a small number of target genes • PAX3-FOXO1 regulates RNA polymerase II pause release at regulated genes • PAX3-FOXO1 controls co-regulatory complex assembly at regulated enhancers Zhang et al. use rapid protein degradation to define the mechanism of transcriptional control by, PAX3-FOXO1, a driver of aRMS. PAX3-FOXO1 functions at a limited number of enhancers to maintain chromatin accessibility and assemble critical co-regulatory complexes to trigger RNA polymerase II pause release and drive target gene expression. [ABSTRACT FROM AUTHOR]

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na+ transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na+-K+-2Cl- cotransporter, NKCC2. [ABSTRACT FROM AUTHOR]

Abstract: Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1–239) that contains one selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240–361) that contains nine selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up by renal proximal convoluted tubule (PCT) cells via megalin-mediated endocytosis. We purified Sepp1 forms from the urine of megalin −/− mice using a monoclonal antibody to the N-terminal domain. Mass spectrometry revealed that the purified urinary Sepp1 consisted of N-terminal fragments terminating at 11 sites between residues 183 and 208. They were therefore designated Sepp1UF. Because the N-terminal domain of Sepp1 has a thioredoxin fold, Sepp1UF were compared with full-length Sepp1, Sepp1Δ240–361, and Sepp1U40S as a substrate of thioredoxin reductase-1 (TrxR1). All forms of Sepp1 except Sepp1U40S, which contains serine in place of the selenocysteine, were TrxR1 substrates, catalyzing NADPH oxidation when coupled with H2O2 or tert-butylhydroperoxide as the terminal electron acceptor. These results are compatible with proteolytic cleavage freeing Sepp1UF from full-length Sepp1, the form that has the role of selenium transport, allowing Sepp1UF to function by itself as a peroxidase. Ultimately, plasma Sepp1UF and small selenium-containing proteins are filtered by the glomerulus and taken up by PCT cells via megalin-mediated endocytosis, preventing loss of selenium in the urine and providing selenium for the synthesis of glutathione peroxidase-3. [Copyright &y& Elsevier]

Staphylococcus aureus causes diseases ranging from superficial wound infections to more invasive manifestations like osteomyelitis and endocarditis. The evasion of host phagocytes recruited to the site of infection is essential to the success of S. aureus as a pathogen. A single S. aureus strain can produce up to five different bicomponent pore-forming leukotoxins that lyse immune cells by forming pores in the cellular plasma membrane. Although these leukotoxins have been considered redundant due to their cytotoxic activity toward human neutrophils, each toxin displays varied species and cell-type specificities. This suggests that cellular factors may influence which cells each toxin targets. Here we describe the identification of CD11b, the a subunit of the αM/β2 integrin (CD11b/CD18), macrophage-1 antigen, or complement receptor 3, as a cellular receptor for leukocidin A/B (LukAB), an important toxin that contributes to S. aureus killing of human neutrophils. We demonstrate that CD11b renders human neutrophils susceptible to LukAB-mediated killing by purified LukAB as well as during S. aureus infection ex vivo. LukAB directly interacts with human CD11b by binding to the I domain, a property that determines the species specificity exhibited by this toxin. Identification of a LukAB cellular target has broad implications for the use of animal models to study the role of LukAB in S. aureus pathogenesis, explains the toxin's tropism toward human neutrophils and other phagocytes, and provides a cellular therapeutic target to block the effect of LukAB toward human neutrophils. [ABSTRACT FROM AUTHOR]

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-d-ribose 5-phosphate (ribose 5-phosphate) and α-d-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator α-d-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn2+-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[18O3]phosphate and [U-13C5]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle. [ABSTRACT FROM AUTHOR]

Cdc14 phosphatases antagonize cyclin-dependent kinase-directed phosphorylation events and are involved in several facets of cell cycle control. We investigate the role of the fission yeast Cdc14 homologue Clp1/Flp1 in cytokinesis. We find that Clp1/Flp1 is tethered at the contractile ring (CR) through its association with anillin-related Mid1. Fluorescent recovery after photobleaching analyses indicate that Mid1, unlike other tested CR components, is anchored at the cell midzone, and this physical properly is likely to account for its scaffolding role. By generating a mutation in mid1 that selectively disrupts Clp1/Flp1 tethering, we reveal the specific functional consequences of Clp1/Flp1 activity at the CR, including dephosphorylation of the essential CR component Cdc15, reductions in CR protein mobility, and CR resistance to mild perturbation. Our evidence indicates that Clp1/Flp1 must interact with the Mid1 scaffold to ensure the fidelity of Schizosaccharomyces pombe cytokinesis. [ABSTRACT FROM AUTHOR]

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes. [Copyright &y& Elsevier]

We have conducted a proteomic analysis of the 70 S ribosome from the Chlamydomonas reinhardtii chloroplast. Twenty-seven orthologs of Escherichia coli large subunit proteins were identified in the 50 S subunit, as well as an ortholog of the spinach plastid-specific ribosomal protein-6. Several of the large subunit proteins of C. reinhardtii have short extension or insertion sequences, but overall the large subunit proteins are very similar to those of spinach chloroplast and E. coli. Two proteins of 38 and 41 kDa, designated RAP38 and RAP41, were identified from the 70 S ribosome that were not found in either of the ribosomal subunits. Phylogenetic analysis identified RAP38 and RAP41 as paralogs of spinach CSP41, a chloroplast RNA-binding protein with endoribonuclease activity. Overall, the chloroplast ribosome of C. reinhardtii is similar to those of spinach chloroplast and E. coli, but the C. reinhardtii ribosome has proteins associated with the 70 S complex that are related to non-ribosomal proteins in other species. In addition, the 30 S subunit contains unusually large orthologs of E. coli S2, S3, and S5 and a novel S1-type protein (Yamaguchi, K. et al., (2002) Plant Cell 14, 29572974). These additional proteins and domains likely confer functions used to regulate chloroplast translation in C. reinhardtii. [ABSTRACT FROM AUTHOR]

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins. [Display omitted] • Human E1 mAbs recognize the conserved fusion loop for pan-alphavirus reactivity • Cryptic E1 epitopes depend on acidic pH for exposure • Human E1 mAbs can inhibit virus egress • Egress inhibition serves as a correlate of protection for EEEV-induced disease Broadly reactive alphavirus E1 antibodies obtained from survivors of natural Eastern equine encephalitis virus infection inhibit virus egress in vitro and protect against infection by encephalitic (Eastern equine encephalitis) and arthritogenic (chikungunya) alphaviruses in mice. [ABSTRACT FROM AUTHOR]

The primary interactions between incoming viral RNA genomes and host proteins are crucial to infection and immunity. Until now, the ability to study these events was lacking. We developed viral cross-linking and solid-phase purification (VIR-CLASP) to characterize the earliest interactions between viral RNA and cellular proteins. We investigated the infection of human cells using Chikungunya virus (CHIKV) and influenza A virus and identified hundreds of direct RNA-protein interactions. Here, we explore the biological impact of three protein classes that bind CHIKV RNA within minutes of infection. We find CHIKV RNA binds and hijacks the lipid-modifying enzyme fatty acid synthase (FASN) for pro-viral activity. We show that CHIKV genomes are N6-methyladenosine modified, and YTHDF1 binds and suppresses CHIKV replication. Finally, we find that the innate immune DNA sensor IFI16 associates with CHIKV RNA, reducing viral replication and maturation. Our findings have direct applicability to the investigation of potentially all RNA viruses. • VIR-CLASP identifies host protein interactions with incoming RNA viral genomes • VIR-CLASP can reveal the early interactomes of seven different viral families • The CHIKV genome binds to distinct proteins under different conditions and times • (Non)canonical RBPs like FASN, IFI16, and YTHDF1 uniquely regulate CHIKV replication The earliest interactions between incoming RNA viral genomes and host RNA-binding proteins can be crucial to viral replication. Until now, these events could not be distinctly studied. Kim et al. develop VIR-CLASP, demonstrate its extensibility across multiple viral families, and report their findings on the CHIKV and IAV interactomes. [ABSTRACT FROM AUTHOR]

Helicobacter pylori infection and a high salt diet are each risk factors for gastric cancer. In this study, we tested the hypothesis that environmental salt concentration influences the composition of the H. pylori exoproteome. H. pylori was cultured in media containing varying concentrations of sodium chloride, and aliquots were fractionated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified proteins that were selectively released into the extracellular space, and we identified selectively released proteins that were differentially abundant in culture supernatants, depending on the environmental salt concentration. We also used RNA-seq analysis to identify genes that were differentially expressed in response to environmental salt concentration. The salt-responsive proteins identified by proteomic analysis and salt-responsive genes identified by RNA-seq analysis were mostly non-concordant, but the secreted toxin VacA was salt-responsive in both analyses. Western blot analysis confirmed that VacA levels in the culture supernatant were increased in response to high salt conditions, and quantitative RT-qPCR experiments confirmed that vacA transcription was upregulated in response to high salt conditions. These results indicate that environmental salt concentration influences the composition of the H. pylori exoproteome, which could contribute to the increased risk of gastric cancer associated with a high salt diet. Helicobacter pylori -induced alterations in the gastric mucosa have been attributed, at least in part, to the actions of secreted H. pylori proteins. In this study, we show that H. pylori growth in high salt concentrations leads to increased levels of a secreted VacA toxin. Salt-induced alterations in the composition of the H. pylori exoproteome is relevant to the increased risk of gastric cancer associated with consumption of a high salt diet. Unlabelled Image • We define effects of salt concentration on the Helicobacter pylori exoproteome. • We define effects of salt concentration on the H. pylori transcriptome. • Levels of VacA toxin increase in response to high salt concentrations. • vacA transcription is upregulated in response high salt concentrations. • The findings help to explain how a high salt diet influences stomach cancer risk. [ABSTRACT FROM AUTHOR]

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2., Competing Interests: Competing interests statement:A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, and Pfizer, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, and Astrazeneca. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York.

The Helicobacter pylori Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from Δ cagT or Δ cagM mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagY ∆ AP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC., (© 2024 Roberts et al.)

Helicobacter pylori colonization of the human stomach is a strong risk factor for gastric cancer. To investigate H. pylori -induced gastric molecular alterations, we used a Mongolian gerbil model of gastric carcinogenesis. Histologic evaluation revealed varying levels of atrophic gastritis (a premalignant condition characterized by parietal and chief cell loss) in H. pylori -infected animals, and transcriptional profiling revealed a loss of markers for these cell types. We then assessed the spatial distribution and relative abundance of proteins in the gastric tissues using imaging mass spectrometry and liquid chromatography with tandem mass spectrometry. We detected striking differences in the protein content of corpus and antrum tissues. Four hundred ninety-two proteins were preferentially localized to the corpus in uninfected animals. The abundance of 91 of these proteins was reduced in H. pylori -infected corpus tissues exhibiting atrophic gastritis compared with infected corpus tissues exhibiting non-atrophic gastritis or uninfected corpus tissues; these included numerous proteins with metabolic functions. Fifty proteins localized to the corpus in uninfected animals were diffusely delocalized throughout the stomach in infected tissues with atrophic gastritis; these included numerous proteins with roles in protein processing. The corresponding alterations were not detected in animals infected with a H. pylori ∆ cagT mutant (lacking Cag type IV secretion system activity). These results indicate that H. pylori can cause loss of proteins normally localized to the gastric corpus as well as diffuse delocalization of corpus-specific proteins, resulting in marked changes in the normal gastric molecular partitioning into distinct corpus and antrum regions.IMPORTANCEA normal stomach is organized into distinct regions known as the corpus and antrum, which have different functions, cell types, and gland architectures. Previous studies have primarily used histologic methods to differentiate these regions and detect H. pylori -induced alterations leading to stomach cancer. In this study, we investigated H. pylori -induced gastric molecular alterations in a Mongolian gerbil model of carcinogenesis. We report the detection of numerous proteins that are preferentially localized to the gastric corpus but not the antrum in a normal stomach. We show that stomachs with H. pylori- induced atrophic gastritis (a precancerous condition characterized by the loss of specialized cell types) exhibit marked changes in the abundance and localization of proteins normally localized to the gastric corpus. These results provide new insights into H. pylori -induced gastric molecular alterations that are associated with the development of stomach cancer., Competing Interests: The authors declare no conflict of interest.

Collagen IV is an essential structural protein in all metazoans. It provides a scaffold for the assembly of basement membranes, a specialized form of extracellular matrix, which anchors and signals cells and provides microscale tensile strength. Defective scaffolds cause basement membrane destabilization and tissue dysfunction. Scaffolds are composed of α-chains that coassemble into triple-helical protomers of distinct chain compositions, which in turn oligomerize into supramolecular scaffolds. Chloride ions mediate the oligomerization via NC1 trimeric domains, forming an NC1 hexamer at the protomer-protomer interface. The chloride concentration-"chloride pressure"-on the outside of cells is a primordial innovation that drives the assembly and dynamic stabilization of collagen IV scaffolds. However, a Cl-independent mechanism is operative in Ctenophora, Ecdysozoa, and Rotifera, which suggests evolutionary adaptations to environmental or tissue conditions. An understanding of these exceptions, such as the example of Drosophila, could shed light on the fundamentals of how NC1 trimers direct the oligomerization of protomers into scaffolds. Here, we investigated the NC1 assembly of Drosophila. We solved the crystal structure of the NC1 hexamer, determined the chain composition of protomers, and found that Drosophila adapted an evolutionarily unique mechanism of scaffold assembly that requires divalent cations. By studying the Drosophila case we highlighted the mechanistic role of chloride pressure for maintaining functionality of the NC1 domain in humans. Moreover, we discovered that the NC1 trimers encode information for homing protomers to distant tissue locations, providing clues for the development of protein replacement therapy for collagen IV genetic diseases., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In vascular smooth muscle cells (VSMCs), LRRC8A volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A physically associates with NADPH oxidase 1 (Nox1) and supports its production of extracellular superoxide (O 2 -• )., Methods and Results: Mice lacking LRRC8A exclusively in VSMCs (Sm22α-Cre, KO) were used to assess the role of VRACs in TNFα signaling and vasomotor function. KO mesenteric vessels contracted normally to KCl and phenylephrine, but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). 48 hours of ex vivo exposure to TNFα (10ng/ml) markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 μM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 35 proteins that interacted with LRRC8A. Pathway analysis revealed actin cytoskeletal regulation as the most closely associated function of these proteins. Among these proteins, the Myosin Phosphatase Rho-Interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots which revealed LRRC8A binding at the second Pleckstrin Homology domain of MPRIP. siLRRC8A or CBX treatment decreased RhoA activity in cultured VSMCs, and MYPT1 phosphorylation at T853 was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure., Conclusions: Interaction of Nox1/LRRC8A with MPRIP/RhoA/MYPT1/actin may allow redox regulation of the cytoskeleton and link Nox1 activation to both inflammation and vascular contractility.

Topological stress can cause converging replication forks to stall during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome fork stalling, suggesting that alternative mechanisms of termination exist. Using proteomics in Xenopus egg extracts, we show that the helicase RTEL1 and the replisome protein MCM10 are highly enriched on chromatin during fork convergence and are crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 cooperate to promote fork convergence and do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 play a general role in promoting progression of stalled forks, including when forks stall during termination. Our data reveal an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Azole drugs represent the primary means of treating infections associated with the filamentous fungal pathogen Aspergillus fumigatus. A central player in azole resistance is the Zn 2 Cys 6 zinc cluster-containing transcription factor AtrR. This factor stimulates expression of both the cyp51A gene, which encodes the azole drug target enzyme, as well as an ATP-binding cassette transporter-encoding gene called abcG1 ( cdr1B ). We used a fusion protein between AtrR and the tandem affinity purification (TAP) moiety to purify proteins that associated with AtrR from A. fumigatus. Protein fractions associated with AtrR-TAP were subjected to multidimensional protein identification technology mass spectrometry, and one of the proteins identified was encoded by the AFUA_6g08010 gene. We have designated this protein NcaA (for nuclear coactivator of AtrR). Loss of ncaA caused a reduction in voriconazole resistance and drug-induced abcG1 expression, although it did not impact induction of cyp51A transcription. We confirmed the association of AtrR and NcaA by coimmunoprecipitation from otherwise-wild-type cells. Expression of fusion proteins between AtrR and NcaA with green fluorescent protein allowed determination that these two proteins were localized in the A. fumigatus nucleus. Together, these data support the view that NcaA is required for nuclear gene transcription controlled by AtrR. IMPORTANCE Aspergillus fumigatus is a major filamentous fungal pathogen in humans and is susceptible to the azole antifungal class of drugs. However, loss of azole susceptibility has been detected with increasing frequency in the clinic, and infections associated with these azole-resistant isolates have been linked to treatment failure and worse outcomes. Many of these azole-resistant strains contain mutant alleles of the cyp51A gene, which encodes the azole drug target. A transcription factor essential for cyp51A gene transcription has been identified and designated AtrR. AtrR is required for azole-inducible cyp51A transcription, but we know little of the regulation of this transcription factor. Using a biochemical approach, we identified a new protein called NcaA that is involved in regulation of AtrR at certain target gene promoters. Understanding the mechanisms controlling AtrR function is an important goal in preventing or reversing azole resistance in this pathogen.

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes., Competing Interests: The authors declare no competing interest.

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read 'Department of Biochemistry and Molecular Genetics and Breast Surgery, Ehime University Graduate School of Medicine, 7910295, Japan' and affiliation 3 incorrectly read 'Department of Hepato-Biliary-Pancreatic and Breast Surgery, Ehime University Graduate School of Medicine, Matsuyama 7910295, Japan.' These errors have now been corrected in both the PDF and HTML versions of the Article.

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene-editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that can associate independently with vesicles and plasma membrane and are in dynamic equilibrium with octamer and monomers. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of SEC3 occurs prior to fusion, whereas other subunits depart just after fusion. About 9 exocyst complexes are associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important insights into assembly/disassembly mechanisms.

Purpose: Helicobacter pylori infection and a high dietary salt intake are each risk factors for the development of gastric cancer. We hypothesize that changes in environmental salt concentrations lead to alterations in the H. pylori membrane proteome., Experimental Design: Label-free and iTRAQ methods were used to identify H. pylori proteins that change in abundance in response to alterations in environmental salt concentrations. In addition, we biotinylated intact bacteria that were grown under high- or low-salt conditions, and thereby analyzed salt-induced changes in the abundance of surface-exposed proteins., Results: Proteins with increased abundance in response to high salt conditions included CagA, the outer membrane protein HopQ, and fibronectin domain-containing protein HP0746. Proteins with increased abundance in response to low salt conditions included VacA, two VacA-like proteins (ImaA and FaaA), outer-membrane iron transporter FecA3, and several proteins involved in flagellar activity. Consistent with the proteomic data, bacteria grown in high salt conditions exhibited decreased motility compared to bacteria grown in lower salt conditions., Conclusion and Clinical Relevance: Alterations in the H. pylori membrane proteome in response to high salt conditions may contribute to the increased risk of gastric cancer associated with a high salt diet., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

The proteome informatics research group of the Association of Biomolecular Resource Facilities conducted a study to assess the community's ability to detect and characterize peptides bearing a range of biologically occurring post-translational modifications when present in a complex peptide background. A data set derived from a mixture of synthetic peptides with biologically occurring modifications combined with a yeast whole cell lysate as background was distributed to a large group of researchers and their results were collectively analyzed. The results from the twenty-four participants, who represented a broad spectrum of experience levels with this type of data analysis, produced several important observations. First, there is significantly more variability in the ability to assess whether a results is significant than there is to determine the correct answer. Second, labile post-translational modifications, particularly tyrosine sulfation, present a challenge for most researchers. Finally, for modification site localization there are many tools being employed, but researchers are currently unsure of the reliability of the results these programs are producing.

Both DNA and chromatin need to be duplicated during each cell division cycle. Replication happens in the context of defects in the DNA template and other forms of replication stress that present challenges to both genetic and epigenetic inheritance. The replication machinery is highly regulated by replication stress responses to accomplish this goal. To identify important replication and stress response proteins, we combined isolation of proteins on nascent DNA (iPOND) with quantitative mass spectrometry. We identified 290 proteins enriched on newly replicated DNA at active, stalled, and collapsed replication forks. Approximately 16% of these proteins are known replication or DNA damage response proteins. Genetic analysis indicates that several of the newly identified proteins are needed to facilitate DNA replication, especially under stressed conditions. Our data provide a useful resource for investigators studying DNA replication and the replication stress response and validate the use of iPOND combined with mass spectrometry as a discovery tool.

In vivo studies have shown that selenium is supplied to testis and brain by apoER2-mediated endocytosis of Sepp1. Although cultured cell lines have been shown to utilize selenium from Sepp1 added to the medium, the mechanism of uptake and utilization has not been characterized. Rat L8 myoblast cells were studied. They took up mouse Sepp1 from the medium and used its selenium to increase their glutathione peroxidase (Gpx) activity. L8 cells did not utilize selenium from Gpx3, the other plasma selenoprotein. Neither did they utilize it from Sepp1(Δ240-361), the isoform of Sepp1 that lacks the selenium-rich C-terminal domain. To identify Sepp1 receptors, a solubilized membrane fraction was passed over a Sepp1 column. The receptors apoER2 and Lrp1 were identified in the eluate by mass spectrometry. siRNA experiments showed that knockdown of apoER2, but not of Lrp1, inhibited (75)Se uptake from (75)Se-labeled Sepp1. The addition of protamine to the medium or treatment of the cells with chlorate also inhibited (75)Se uptake. Blockage of lysosome acidification did not inhibit uptake of Sepp1 but did prevent its digestion and thereby utilization of its selenium. These results indicate that L8 cells take up Sepp1 by an apoER2-mediated mechanism requiring binding to heparin sulfate proteoglycans. The presence of at least part of the selenium-rich C-terminal domain of Sepp1 is required for uptake. RT-PCR showed that mouse tissues express apoER2 in varying amounts. It is postulated that apoER2-mediated uptake of long isoform Sepp1 is responsible for selenium distribution to tissues throughout the body.

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function.

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

Affinity isolation of protein complexes followed by protein identification by LC-MS/MS is an increasingly popular approach for mapping protein interactions. However, systematic and random assay errors from multiple sources must be considered to confidently infer authentic protein-protein interactions. To address this issue, we developed a general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes' Odds estimation. We then applied our LRT-Bayes' algorithm experimentally using data from protein complexes isolated from Rhodopseudomonas palustris. Our algorithm, in conjunction with the experimental protocol, inferred with high confidence authentic interacting proteins from abundant, stable complexes, but few or no authentic interactions for lower-abundance complexes. The algorithm can discriminate against a background of prey proteins that are detected in association with a large number of baits as an artifact of the measurement. We conclude that the experimental protocol including the LRT-Bayes' algorithm produces results with high confidence but moderate sensitivity. We also found that Monte Carlo simulation is a feasible tool for checking modeling assumptions, estimating parameters, and evaluating the significance of results in protein association studies.

The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum. Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

Proteomics is providing us with a variety of exciting new strategies to address biological questions. These strategies must integrate a series of seperative and analytical technologies to deal with the immense complexity involved. At some point in the process, the proteins are usually digested with a proteolytic enzyme to generate shorter peptides that are more easily analyzed by mass spectrometry. Shotgun proteomics relies on separation after this digestion step and takes advantage of tandem mass spectrometry to infer the amino acid sequence of individual peptides. Advances in quantitation, and the ability to find sites of post-translational modification are expanding the scope of questions that can be asked. The ultimate success of any proteomic experiment is dictated not only by an appropriate choice of seperative and analytical techniques, but also by making certain that the biological aspects of the experiment are focused and well designed.

To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.

The 26S proteasome mediates degradation of ubiquitin-conjugated proteins. Although ubiquitin is recycled from proteasome substrates, the molecular basis of deubiquitination at the proteasome and its relation to substrate degradation remain unknown. The Rpn11 subunit of the proteasome lid subcomplex contains a highly conserved Jab1/MPN domain-associated metalloisopeptidase (JAMM) motif-EX(n)HXHX(10)D. Mutation of the predicted active-site histidines to alanine (rpn11AXA) was lethal and stabilized ubiquitin pathway substrates in yeast. Rpn11(AXA) mutant proteasomes assembled normally but failed to either deubiquitinate or degrade ubiquitinated Sic1 in vitro. Our findings reveal an unexpected coupling between substrate deubiquitination and degradation and suggest a unifying rationale for the presence of the lid in eukaryotic proteasomes.

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.