Proteomic identification of Listeria monocytogenes surface‐associated proteins

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano‐LC and online‐MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy‐four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface‐exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.     

Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a well‐known probiotic bacterium extensively studied for its beneficial health effects. Exoproteome (proteins exported into culture medium) and surface proteome (proteins attached to S‐layer) of this probiotic were identified by using 2DE followed by MALDI TOF MS to find proteins potentially involved in bacteria–host interactions. The exo‐ and surface proteomes included 43 and 39 different proteins from 72 and 49 successfully identified spots, respectively. Twenty‐two proteins were shared between the two proteomes; both contained the major surface layer protein that participates in host interaction as well as several well‐known and putative moonlighting proteins. The exoproteome contained nine classically‐secreted (containing a signal sequence) and ten nonclassically‐secreted proteins, while the surface proteome contained four classically‐secreted and eight nonclassically secreted proteins. Identification of exo‐ and surface proteomes contributes describing potential protein‐mediated probiotic–host interactions.     

MS‐based ligand binding assays with speed,sensitivity, and specificity

Immunoassays are widely used in biochemical/clinical laboratories owing to their simplicity, speed, and sensitivity. We combined self‐assembled monolayer‐based immunoassays with MALDI‐TOF MS to show that high‐fidelity surface preparations with a novel matrix deposition/crystallization technique permits quantitative analysis of monolayer‐bound antigens at picomolar detection limits. Calibration curves for intact proteins are possible over a broad concentration range and improved specificity of MS‐immunoassays is highlighted by simultaneous label‐free quantitation of ligand‐bound protein complexes.     

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

The barnacle, Balanus amphitrite, is one of the primary model organisms for rocky-shore ecology studies and biofouling research. This barnacle species has a complex life cycle during which the swimming nauplius molts six times and transforms into a cyprid stage. Cyprids must attach to a surface to metamorphose into a juvenile barnacle. To clarify the overall profile of protein expression during larval development and metamorphosis, 2-DE was used to compare the proteome of the nauplius, the swimming cyprid, the attached cyprid, and the metamorphosed cyprid. The proteome of the swimming cyprid was distinctly different from that of other life stages and had about 400 spots. The proteomes of the attached and metamorphosed cyprids were similar with respect to major proteins but had significantly lower numbers of spots compared to that of swimming larval stages. Obviously, synthesis of most proteins from swimming cyprids was switched off after attachment and metamorphosis. Our advanced MS analysis (MALDI-TOF/TOF MS/MS) allowed us to identify the proteins that were differentially and abundantly expressed in the swimming cyprid. These proteins included signal transduction proteins (adenylate cyclase and calmodulin) and juvenile hormone binding proteins. In summary, for the first time, we have analyzed the global protein expression pattern of fouling marine invertebrate larvae during metamorphosis. Our study provides new insights into the mechanisms of barnacle larval metamorphosis and also provides a foundation for exploring novel targets for antifouling treatments.     

Identification of shed proteins from Chinese hamster ovary cells: application of statistical confidence using human and mouse protein databases

The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation to develop a fundamental understanding of the bystander response. Chinese hamster ovary cells were chosen because they have been widely used for radiation studies and are reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and Fourier transform-ion cyclotron resonance (FT-ICR)-mass spectrometry (MS) analyses. Since the hamster genome has not been sequenced, MS data was searched against the mouse and human protein databases. Nearly 150 proteins identified by tandem mass spectrometry were confirmed by FT-ICR. When both types of MS data were evaluated, using a new confidence scoring tool based on discriminant analyses, about 500 proteins were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface and, hence were likely shed. However, estimates of quantitative changes, based on two independent MS approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using MS in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.     

Detection and identification of protein variants and adducts in blood and tissues: an application of soft ionization mass spectrometry to clinical diagnosis

The detection and identification of protein variants and abnormally increased modified proteins are important for clinical diagnosis. We applied soft ionization mass spectrometry (MS) to analyze proteins in blood and tissues from various patients. Over the past 8 years, we diagnosed 132 cases (55 kinds) of variant proteins including hemoglobin (Hb), transthyretin (TTR), and Cu/Zn-superoxide dismutase (SOD-1), using MS as the leading technology. Of these variants, eight were new, and nine were the first cases in Japan. Some abnormal Hb cause diseases, and most of them cause erroneous levels of glycated Hb, HbA1c, i.e., a popular index of diabetes. Most of the variant TTR causes amyloidotic polyneuropathy. Variant SOD-1 causes amyotrophic lateral sclerosis. We first showed that immunoprecipitation by a specific antiserum is a reliable and simple method to prepare protein from sera and tissues for analysis by matrix-assisted laser desorption time-of-flight MS, and liquid chromatography-electrospray ionization MS (LC-ESI-MS). The use of this technology has become widespread. Using an immunoprecipitated target protein and LC-ESI-MS, we showed that the ratios of tetra-, di- and a-sialo-transferrin from two cases of congenital glycoprotein deficient syndrome were clearly distinguishable from those of control samples. We first reported a unique modified form of TTR, that is, S-sulfonated TTR, which increased markedly and specifically in three cases with molibdenum cofactor deficiency. We proposed that S-sulfonated TTR is a useful marker for screening this disease. ESI-MS was successfully used for the accurate determination of HbA1c, and we clarified the extent of discrepancies between the HbA1c value measured by conventional methods and the accurate values for samples containing various Hb variants determined by the MS method.     

Shedding & shaving: disclosure of proteomic expressions on a bacterial face

Bacterial surface proteins are critically important in determining the success of bacterial strains in their competition for survival, which makes comprehensive knowledge of the microbial 'face' of high relevance to understand bacterial physiology. Therefore, the aim of this study was to inventorize the proteomic composition of a bacterial surface by gel-free approaches using Bacillus subtilis as a model organism. To this purpose, intact bacteria were incubated with trypsin to cleave surface proteins, after which these 'shaved' proteins were identified by LC-MS/MS analysis of tryptic peptides that were released into the incubation buffer. In parallel, proteins that were released from bacterial cells without trypsin treatment were inventorized and defined as 'shed' proteins. The results showed that peptides of 41 proteins, comprising transmembrane proteins, secretory and lipoproteins, known 'anchorless' surface proteins and ribosomal proteins, were specifically released by shaving. Some of these proteins were also cleaved by trypsin-beads, which are unable to penetrate the bacterial cell wall, indicating a genuine surface-exposed localization. Together this shows that these combined gel-free approaches can reveal novel proteomic decorations on a bacterial face and may provide leads for future studies on protein sorting in B. subtilis and other Gram-positive bacteria.     

Nanozeolite‐driven approach for enrichment of secretory proteins in human hepatocellular carcinoma cells

Given the importance of secreted proteins as a source for early detection and diagnosis of disease, secreted proteins have been arousing considerable attention. However, the analysis of secreted proteins represents a challenge for current proteomic techniques. One of the difficulties in secretomic study is to concentrate proteins from large volume of growth media, particularly, the low abundant and low molecular weight proteins (molecular weight <30 kDa). Herein, we describe a novel strategy for harvesting secretory proteins. In this approach, proteins secreted from the human hepatocellular carcinoma cell line were enriched by zeolite LTL nanocrystals, followed by 1‐D SDS‐PAGE for protein fractionation and then by LC‐ESI‐MS/MS for protein identification. In total, 1474 unique proteins were confidently identified, including 505 low molecular weight proteins, and covered a broad range of pI and molecular weight. Furthermore, this study not only offered an efficient and powerful method for the enrichment of secretory proteins but also allowed in‐depth study of secretome of hepatocellular carcinoma cells. The reported work is expected to represent one of the most comprehensive secretomic analyses so far.     

Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification and differential protein expression

Proteomic analyses of the proliferative and secretory phases of the human endometrium were carried out to identify proteins and discover differentially expressed proteins using isotope-coded affinity tags, three stages of chromatographic separation and online tandem mass spectrometry (MS/MS). From an initial list of 346 proteins identified by ProICAT, manual inspection of MS/MS spectra and confirmatory searches pared the list down to 119 positively identified proteins. Only five of the proteins showed consistent differential expression. The utility of some of these proteins as indicators of true differential expression in the endometrium is open to discussion. The two proteins with unquestionable differential expressions in the secretory endometrium are: glutamate NMDA receptor subunit zeta 1 precursor and FRAT1. Some of the proteins that show no differential expression have previously been examined in gene-expression studies with similar conclusions.     

Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.     

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.     

Fluorescence microscopy of the dynamics of supercoiling, folding, and condensation of bacterial chromosomes, induced by acridine orange

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and supercoiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA, and indicate that kinks are an important intermediate step in many of the transitions.     

A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.     

Protein interactions with the platelet integrin αIIb regulatory motif

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin α‐subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet‐specific integrin α_IIbβ₃. We employ peptide‐affinity chromatography followed‐up with LC‐MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR‐peptide. Of these, seven have been reported in the PPI literature as interactors with integrin α‐subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin‐tagged α‐integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide‐affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.     

Isolation,culture, and characterization of equine oviduct epithelial cells in vitro

Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17β or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers. © 1995 Wiley-Liss, Inc.     

Fluorescence Microscopy of the Dynamics of Supercoiling,Folding, and Condensation of Bacterial Chromosomes,Induced by Acridine Orange

Abstract

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and super- coiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA and indicate that kinks are an important intermediate step in many of the transitions.     

Proteome analysis of embryo and endosperm from germinating tomato seeds

Proteome analysis of embryo and endosperm tissues from germinating tomato seed was conducted using 1-DE, 2-DE, and MS. Mobilization of the most abundant proteins, which showed similar profiles in the two tissues, occurred first in the endosperm. CBB R-250 staining of 2-DE gels revealed 352 and 369 major protein spots in the embryo and endosperm, respectively, at 0 h. Of these, 75 major spots were selected, excised, in-gel digested with trypsin, and analyzed by MALDI-TOF-MS and/or LC-ESI-Q/TOF-MS/MS. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 47 proteins identified. Embryo-specific proteins included a BAC19.13 homologue, whereas four proteins specific to the endosperm were tomato mosaic virus coat proteins related to defense mechanisms. The most abundant proteins both in the embryo and endosperm were seed storage proteins, i.e., legumins (11 spots), vicilins (11 spots), albumin (2 spots). Housekeeping enzymes, actin-binding profilin, defense-related protein kinases, nonspecific lipid transfer protein, and proteins involved in general metabolism were also identified. The roles of some of the proteins identified in the embryo and endosperm are discussed in relation to seed germination in tomato.     

Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide

Osseointegration is the structural and functional connection between bone tissues and implants such as titanium dioxide (TiO₂). The bone-TiO₂ interface is thought to contain proteoglycans. However, exhaustive analysis of the proteins in this layer has not been performed. In this study, we evaluated the bone protein adhered on the surface of TiO₂ comprehensively. Pig bone protein was extracted by sequential elutions with guanidine, 0.1 M EDTA, and again with guanidine. The proteins obtained from these extractions were allowed to adhere to an HPLC column packed with TiO₂ and were eluted with 0.2 M NaOH. The eluted proteins were identified by LC/MS/MS and included not only proteoglycans but also other proteins such as extracellular matrix proteins, enzymes, and growth factors. Calcium depositions were observed on TiO₂ particles incubated with bone proteins, guanidine-extracted proteins adhered to TiO₂ displayed significantly high amounts of calcium depositions.     

Differential arrival of newly synthesized apical and basolateral plasma membrane proteins in the epithelial cell line A6

The labeling of specific cell surface proteins with biotin was used to examine both protein distribution and delivery of newly synthesized proteins to the apical and basolateral cell surface in A6 cells. Steady-state metabolic labeling with [³⁵S]methionine followed by specific cell surface biotinylation demonstrated polarization of membrane proteins. The delivery of newly synthesized proteins to the apical or basolateral cell surface was examined by metabolic labeling with [³⁵S]methionine using a pulse-chase protocol in combination with specific cell surface biotinylation. Newly synthesized biotinylated proteins at the apical cell surface reached a maximum after a 5 min chase, and then fell over the remainder of a 2 hr chase. The bulk flow of newly synthesized proteins to the basolateral membrane slowly rose to a maximum after 90 min. The detergent Triton X-114 was used to examine delivery of hydrophilic and hydrophobic proteins to the cell surface. Delivery of both hydrophilic and hydrophobic proteins to the apical cell surface reached a maximum 5 to 10 min into the chase period. The arrival of hydrophilic proteins at the basolateral surface showed early delivery and a maximum peak delivery at 120 min into the chase period. In contrast, only an early peak of delivery of newly synthesized hydrophobic proteins to the basolateral membrane was observed.This work was supported by grants from the American Heart Association, the National Kidney Foundation of the Delaware Valley, and from the Department of Veterans Affairs. T.R.K. is a recipient of an Established Investigatorship Award from the American Heart Association.