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Biofilms composed of multiple microorganisms colonize the surfaces of indwelling urethral catheters that are used serially by neurogenic bladder patients and cause chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, and Enterococcus spp., species that cycle through adhesion and multilayered cell growth, trigger host immune responses, are starved off nutrients, and then disperse. Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, promising new insights into this pathology based on host and microbial meta-omics analyses from clinical specimens. We examined catheters from nine neurogenic bladder patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene sequencing and liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic drug treatments of patients resulted in either transient or lasting microbial community perturbations. Neutrophil effector proteins were abundant not only in UP but also CB samples, indicating their penetration of biofilm surfaces. In the context of one patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested a combination of factors associated with this disease complication: CB biomasses were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory systems more than in CBs from other patients. The NADH:quinone oxidoreductase II (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate reductase A (Nar) equipped the pathogen with electron transport chains promoting growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of transition metal ion acquisition systems in response to human host-mediated iron and zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated.

3-M primordial dwarfism is an inherited disease characterized by severe pre- and postnatal growth retardation and by mutually exclusive mutations in three genes, CUL7, OBSL1, and CCDC8. The mechanism underlying 3-M dwarfism is not clear. We showed here that CCDC8, derived from a retrotransposon Gag protein in placental mammals, exclusively localized on the plasma membrane and was phosphorylated by CK2 and GSK3. Phosphorylation of CCDC8 resulted in its binding first with OBSL1, and then CUL7, leading to the membrane assembly of the 3-M E3 ubiquitin ligase complex. We identified LL5β, a plasma membrane protein that regulates cell migration, as a substrate of 3-M ligase. Wnt inhibition of CCDC8 phosphorylation or patient-derived mutations in 3-M genes disrupted membrane localization of the 3-M complex and accumulated LL5β. Deletion of Ccdc8 in mice impaired trophoblast migration and placental development, resulting in intrauterine growth restriction and perinatal lethality. These results identified a mechanism regulating cell migration and placental development that underlies the development of 3-M dwarfism.

Summary The kidney local microenvironment (KLM) plays a critical role in the pathogenesis of kidney fibrosis. However, the composition and regulation of a fibrotic KLM remain unclear. Through a multidisciplinary approach, we investigated the roles of the hepatocyte growth factor/c-met signaling pathway in regulating KLM formation in various chronic kidney disease (CKD) models. We performed a retrospective analysis of single-cell RNA sequencing data and determined that tubular epithelial cells and macrophages are two major cell populations in a fibrotic kidney. We then created a mathematical model that predicted loss of c-met in tubular cells would cause greater responses to injury than loss of c-met in macrophages. By generating c-met conditional knockout mice, we validated that loss of c-met influences epithelial plasticity, myofibroblast activation, and extracellular matrix synthesis/degradation, which ultimately determined the characteristics of the fibrotic KLM. Our findings open the possibility of designing effective therapeutic strategies to retard CKD., Graphical abstract, Highlights • A fibrotic kidney local microenvironment (KLM) is focal and segmental • C-met is more enriched in the distal tubule and collecting duct than in macrophages • Loss of c-met in tubular cells causes superior quick responses to simulated injury • HGF/c-met pathway determines the characteristics of a formed fibrotic KLM

Neutrophils are the most abundant type of white blood cells in humans with biological roles relevant to inflammation and fighting infections. The release of neutrophil extracellular DNA aims to control invasion by bacteria, viruses, fungi, and tissue damage. Neutrophil Extracellular Traps (NETs) act as antimicrobial agents triggering immune signaling through the release of the nuclear content into the extracellular space. Although intense investigations have elucidated the pathways preceding NET formation, the exact molecular composition of released NETs has not been mapped. We aimed to decode the sequences of DNA and proteins from NETs. With emerging needs to understand neutrophil functions precisely, we open the field of NETOMIC studies through isolation of NETs in combination with omics approaches including shotgun genomics and proteomics. Our in vitro NET isolation methodology allowed for unprecedented replicability with induction in a sterile inflammation model system. Enrichment of mitochondrial DNA and telomere sequences are significantly expressed in NET genomes. This study revealed that the genomic sequence released in the extracellular milieu is not stochastically serving as a scaffold for a repertoire of proteins involved in neutrophil protective functions. Collectively, we established the gene and protein signatures exclusive to the extracellular NETs in comparison to undifferentiated and differentiated neutrophil states, further guiding future detection of specific regions needed for diagnostics and targeted therapies of NET related conditions.

Neutrophils are important mediators of the antimicrobial defense during urinary tract infections (UTIs). When activated at the site of infection, these innate immune cells phagocytose and neutralize an invading pathogen. Another neutrophil defense strategy is the release of effectors, such as antimicrobial peptides and proteins stored in neutrophil granules and reactive oxygen species. Their release can be facilitated by cellular signals that trigger chromatic decondensation and the disruption of nuclear membranes, followed by granule and plasma membrane disintegration, DNA release into the extracellular milieu, and neutrophil cell death. Neutrophil extracellular traps (NETs) form. If microbial pathogens are the cause of neutrophil infiltration, they are entrapped in the network of DNA fibers that characterize NETs and are exposed to antimicrobial granule effectors and histones that bind to the extracellular DNA fibers. Here, we describe nonmicroscopic methods applied to clinical (urine sediment) samples to identify and characterize NETs associated with UTI. A stepwise extraction procedure using PBS, deoxyribonuclease I digestion and SDS-based solubilization is described. This is followed by native gel analysis to visualize protein-DNA macromolecular assemblies and proteomic analysis to identify signature proteins and their quantities in NETs. Microbes observed to be entrapped in NETs in the process of the innate immune response to the infection are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, and Enterococcus faecalis.

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems.

Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ~10 μg of total protein were processed by 96FASP and LC-MS resulting in 700-900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics.

Aerococcus urinae ( Au) and Globicatella sanguinis ( Gs) are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The Gs and Au proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)–based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the Au vs Gs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in Gs proteomes) and to the enzyme Xfp that degrades d-xylulose-5′-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6′-phosphate and 4′-phosphopantothenate (expressed only in Au proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for Gs while Au expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that Au and Gs fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.

SummaryThe hepatitis B virus (HBV) regulatory protein X (HBx) activates gene expression from the HBV covalently closed circular DNA (cccDNA) genome. Interaction of HBx with the DDB1-CUL4-ROC1 (CRL4) E3 ligase is critical for this function. Using substrate-trapping proteomics, we identified the structural maintenance of chromosomes (SMC) complex proteins SMC5 and SMC6 as CRL4HBx substrates. HBx expression and HBV infection degraded the SMC5/6 complex in human hepatocytes in vitro and in humanized mice in vivo. HBx targets SMC5/6 for ubiquitylation by the CRL4HBx E3 ligase and subsequent degradation by the proteasome. Using a minicircle HBV (mcHBV) reporter system with HBx-dependent activity, we demonstrate that SMC5/6 knockdown, or inhibition with a dominant-negative SMC6, enhance HBx null mcHBV-Gluc gene expression. Furthermore, SMC5/6 knockdown rescued HBx-deficient HBV replication in human hepatocytes. These results indicate that a primary function of HBx is to degrade SMC5/6, which restricts HBV replication by inhibiting HBV gene expression.

Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nm rather than 212 nm at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation.

By using an unambiguous in vivo deuterated-leucine labeling quantitative proteomic approach, at close to the physiologically relevant level, we systematically profiled multiple proteins interacting with 14-3-3epsilon, the isoform with least characterized protein interactions in 14-3-3 family in mammalian cells. Among the 19 proteins interacting with 14-3-3epsilon identified, 6 of them including SKb1Hs, p54nrb, serine/threonine kinase 38, MEP50, 14-3-3theta and cofilin 2 were the previously unknown interacting partners with 14-3-3epsilon. The newly identified interactor cofilin 2 was also validated in co-transfection and co-immunoprecipitation. In contrast, with the same stringent criteria only three known partners were identified by conventional tandem affinity purification (TAP) approach. Therefore the 'in-spectra' quantitative marker of deuterated-leucine assisted to precisely identify those genuine interacting partners with minimum requirement of validation using other molecular approaches.

To improve the efficiency, accuracy, reproducibility, throughput and proteome coverage of mass spectrometry-based quantitative approaches, both in vitro and in vivo tagging of particular amino acid residues of cellular proteins have been introduced to assist mass spectrometry for global-scale comparative studies of differentially expressed proteins/modifications between different biologically relevant cell states or cells at different pathological states. The basic features of these methods introduce pair-wise isotope signals of each individual peptide containing a particular type of tagged amino acid (amino acid-coded mass tagging) that originated from different cell states. In this review, the applications of major amino acid-coded mass tagging-based quantitative proteomics approaches, including isotope-coded affinity tag, isobaric tags for relative and absolute quantification (iTRAQ) and stable isotope labeling by amino acids in cell culture are summarized in the context of their respective strengths/weakness in identifying those differentially expressed or post-translational modified proteins regulated by particular cellular stress on a genomic scale in a high-throughput manner. Importantly, these gel-free, in-spectra quantitative mechanisms have been further explored to identify/characterize large-scale protein-protein interactions involving various functional pathways. Taken together, the information about quantitative proteome changes, including multiple regulated proteins and their interconnected relationships, will provide an important insight into the molecular mechanisms, where novel targets for diagnosis and therapeutic intervention will be identified.

Urine is one of the most important biofluids in clinical proteomics, and in the past decades many potential disease biomarkers have been identified using mass spectrometry-based proteomics. Current studies mainly perform analyses of the urine supernatant devoid of cells and cell debris, and the pellet (or sediment) fraction is discarded. However, the pellet fraction is biologically of interest. It may contain whole human cells shed into the urine from anatomically proximal tissues and organs (e.g., kidney, prostate, bladder, urothelium, and genitals), disintegrated cells and cell aggregates derived from such tissues, viruses and microbial organisms which colonize or infect the urogenital tract. Knowledge of the function, abundance, and tissue of origin of such proteins can explain a pathological process, identify a microbe as the cause of urinary tract infection, and measure the human immune response to the infection-associated pathogen(s). Successful detection of microbial species in the urinary pellet via proteomics can serve as a clinical diagnostic alternative to traditional cell culture-based laboratory tests. Filter-aided sample preparation (FASP) has been widely used in shotgun proteomics. The methodology presented here implements an effective lysis of cells present in urinary pellets, solubilizes the majority of the proteins derived from microbial and human cells, and generates enzymatic digestion-compatible protein mixtures using FASP followed by optimized desalting procedures to provide a peptide fraction for sensitive and comprehensive LC-MS/MS analysis. A highly parallel sample preparation method in 96-well plates to allow scaling up such experiments is discussed as well. Separating peptides by nano-LC in one dimension followed by online MS/MS analysis on a Q-Exactive mass spectrometer, we have shown that more than 1,000 distinct microbial proteins and 1,000 distinct human proteins can be identified from a single experiment.

Two different dispersion methods were studied in order to obtain rhenium[Re-188] sulfide suspension with different particle size distributions. The manufactured swirling device offers Suspension I with larger particles (55%> 5 mu m, 19%> 10 mu m). However, ultrasonication can only produce Suspension II with smaller particles(93% 10 mu m). Stability tests indicated that deposition appears after 6 and 15 minutes for Suspensions I and II, respectively. Radiochemical purity and particle size distribution did not change distinctively within 24 hours. So, both suspensions can be used in animal tests to find out the optimal particle size ranges for intra-articular injection.

Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins., Author Summary Urinary tract infections (UTIs) are one of the world’s most widespread infectious diseases, with an estimated number of 150 million cases per year. Neutrophils play an important role in the defense of human patients against microbes causing UTIs. Molecules produced by neutrophils that migrate into the urinary tract can kill the invading microbes and resolve an infection, often without a need to treat patients with an antibiotic. Our work shows strong support for a mechanism called the formation of neutrophil extracellular traps (NETs), previously described for other infections and autoimmune conditions, which are involved in killing pathogens that have invaded the urinary tract. We show evidence of extracellular chromatin-containing structures using immunofluorescence microscopy and identified proteins that bind to the chromatin DNA and have functions to damage and kill bacterial cells or stop their growth.

Urine has been an important body fluid source for diagnostic and prognostic biomarkers of diseases for a long time. Technological advances during the last two decades have enabled a fundamental shift from the discovery of candidate protein biomarkers using single-assay platforms to highly parallel liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomic analysis platforms. MS/MS-based approaches such as multiple reaction monitoring (MRM) are also being used increasingly for targeted protein biomarker validation. In large part due to the fact that the majority of protein in voided urine is soluble, such studies have focused on the analysis of urine supernatants, whereas the pellets were discarded after centrifugal sedimentation. Urine sediments are of particular value in the analysis of urinary tract infections (UTI). The LC-MS/MS methods now have sufficient resolving power and sensitivity to survey metaproteomes—the entirety of proteins derived from multiple organisms that interact with each other in mutualistic or antagonistic fashion. Challenges of proteomic analysis of urine include the high dynamic range of protein abundance, high levels of protein post-translational modifications, and high quantities of natural protease inhibitors. Recently, a robust and scalable workflow that can parallelize the processing of multiple urinary supernatant and sediment samples was developed and validated in our lab. This method utilizes 96-well format filter-aided sample preparation (96FASP) strategy and was shown to successfully identify large numbers of proteins from urine samples. Processing 10–50 µg total protein in single experiment, LC-MS/MS with a Q-Exactive mass spectrometer resulted in more than 1,100 distinct human protein identifications from urine supernatants, and around 400 microbial and 1,400 human protein identifications from urine sediments. The surveys are a rich data resource not only for biomarker discovery but also to interrogate mechanisms of pathogenesis in the urinary system.

Phosphatidylinositides are one family of the most versatile signaling molecules in cells, yet how they interact with different proteins to regulate biological processes is not well understood. Towards a general strategy to identify phosphatidylinositide-protein interactions, a fluorous diazirine group has been incorporated into phosphatidylinositol 4,5-bisphosphate (PIP(2)). The modified PIP(2) was effectively cleaved by phospholipase C, one signaling protein that utilizes PIP(2) as its endogenous substrate. Upon light illumination, the PIP(2) probe effectively crosslinks with small GTPase ADP-ribosylation 1 to form a complex, suggesting that the probe might be suitable to identify PIP(2)-interacting proteins on the proteome level.

The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. The Consortium is funded by grants from the NHGRI as follows: production grants: U54HG004570 (B. E. Bernstein); U01HG004695 (E. Birney); U54HG004563 (G. E. Crawford); U54HG004557 (T. R. Gingeras); U54HG004555 (T. J. Hubbard); U41HG004568 /n(W. J. Kent); U54HG004576 (R. M. Myers); U54HG004558 (M. Snyder);/nU54HG004592 (J. A. Stamatoyannopoulos). Pilot grants: R01HG003143 (J. Dekker); RC2HG005591 and R01HG003700 (M. C. Giddings); R01HG004456-03 (Y. Ruan); U01HG004571 (S. A. Tenenbaum); U01HG004561 (Z. Weng); RC2HG005679 (K. P. White). This project was supported in part by American Recovery and/nReinvestment Act (ARRA) funds from the NHGRI through grants U54HG004570, U54HG004563, U41HG004568, U54HG004592, R01HG003143, RC2HG005591,R01HG003541, U01HG004561, RC2HG005679andR01HG003988(L. Pennacchio). In addition, work from NHGRI Groups was supported by the Intramural Research/nProgram of the NHGRI (L. Elnitski, ZIAHG200323; E. H. Margulies, ZIAHG200341). Research in the Pennachio laboratory was performed at Lawrence Berkeley National Laboratory and at the United States Department of Energy Joint Genome Institute, Department of Energy Contract DE-AC02-05CH11231, University of California.

To further improve the selectivity and throughput of phosphopeptide analysis for the samples from real-time cell lysates, here we demonstrate a highly efficient method for phosphopeptide enrichment via newly synthesized magnetite microparticles and the concurrent mass spectrometric analysis. The magnetite microparticles show excellent magnetic responsivity and redispersibility for a quick enrichment of those phosphopeptides in solution. The selectivity and sensitivity of magnetite microparticles in phosphopeptide enrichment are first evaluated by a known mixture containing both phosphorylated and nonphosphorylated proteins. Compared with the titanium dioxide-coated magnetic beads commercially available, our magnetite microparticles show a better specificity toward phosphopeptides. The selectively-enriched phosphopeptides from tryptic digests of β-casein can be detected down to 0.4 fmol μl⁻¹, whereas the recovery efficiency is approximately 90% for monophosphopeptides. This magnetite microparticle-based affinity technology with optimized enrichment conditions is then immediately applied to identify all possible phosphorylation sites on a signal protein isolated in real time from a stress-stimulated mammalian cell culture. A large fraction of peptides eluted from the magnetic particle enrichment step were identified and characterized as either single- or multiphosphorylated species by tandem mass spectrometry. With their high efficiency and utility for phosphopeptide enrichment, the magnetite microparticles hold great potential in the phosphoproteomic studies on real-time samples from cell lysates.

Corrigendum to ‘‘An optimized magnetite microparticle-based phosphopeptide enrichment strategy for identifying multiple phosphorylation sites in an immunoprecipitated protein’’ [Anal. Biochem. 408 (2011) 19–31] Yi Huang , Qihui Shi , Chia-Kuang Tsung , Harsha P. Gunawardena , Ling Xie , Yanbao Yu , Hongjun Liang , Pengyuan Yang , Galen D. Stucky c,⇑⇑, Xian Chen a,b,⇑ Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, People’s Republic of China Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA

Selecting reasonable matches from the database search results is crucial to mass spectrometry-based proteomics identification. However, the current score-based filter solution and decoy database methods are not effective enough to prevent all false positive and false negative selections. In this study, a systematic search strategy named iterative non-m/z-sharing (INMZS) analysis was proposed to address the problem. In the strategy, all search results were screened based on the share status of corresponding matched m/z, only the proteins that matched with exclusive m/z information were reserved for the confident matches. The researchers did further iterative search to improve the identification of minor components in a spot. Finally, identifications were confirmed by decoy database evaluation for the final phase of protein identification. Simulation and application tests of INMZS were implemented on a large human liver data set and standard protein cocktails. The result shows that INMZS plus decoy database evaluation is efficient in ensuring the confidence and sensitivity of 2-DE or similar non-shotgun based proteome identification.