Helicobacter pylori proteomics by 2‐DE/MS, 1‐DE‐LC/MS and functional data mining

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea‐solubilized proteins by 2‐DE/MS (data set 2‐DE) and by 1‐DE‐LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1‐DE‐LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research ( http://www.mpiib‐berlin.mpg.de/2D‐PAGE/ ), which gives access to raw mass spectra (MALDI‐TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT ( http://webclu.bio.wzw.tum.de/prompt/ ) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2‐DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2‐DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea‐insoluble proteins and makes them accessible for separation by SDS‐PAGE and LC. The 2‐DE/MS analysis with urea‐solubilized proteins and the 1‐DE‐LC/MS analysis with the urea‐insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT‐generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.     

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.     

Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface‐associated and whole‐cell proteins by two complementary proteomics approaches, 1D‐SDS‐PAGE combined with LC‐ESI‐MS/MS and 2D‐LC‐MALDI‐TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin‐receptor protein for the uptake of siderophore‐bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.     

Cover Picture: Proteomics 5/2009

In this issue of Proteomics you will find the following highlighted articles: Heart (pump) broken? Hearts are pumps within pumps within channels and pumps. Calcium is pumped, potassium, sodium, amino acids, and electrons are all pumped, channeled or driven until, finally, blood is pumped. Failure of one or more pumps leads to a heart attack. This report from Zlatkovic et al. looks at the sub‐proteome associated with hypertensive failure of the K⁺ATP channel and associated cardiomyopathy that develops in KIR6.2 knock‐out mice. Out of >900 reproducible 2‐DE spots, 81 displayed significant over‐ or under‐expression, a number of which validated previously proposed interactions with the Kir6.2 channel. Two‐thirds were down‐regulations, including creatine kinase, adenylate kinase, and lactate dehydrogenase. A total of 114 proteins were ontologically mapped into the K⁺ATP‐dependent sub‐proteome and a role in hypertensive heart failure. Interaction mapping found >240 nodes and >1200 interactions/edges. A good foundation for future work. Zlatkovic, J. et al., Proteomics 2009, 9, 1314‐1325. The deeper you dig, the more you find A classical biochemist interested in protein‐protein interactions purifies his protein away from other proteins, seeking the highest “‐fold purification”. A proteomicist, on the other hand, looks for “consistent contamination” – i.e. association – of the protein of interest with other proteins. This requires high resolution separations and high accuracy concentration determinations. You can only work with species with concentrations above the detection limit (DL) for the detection method. 2‐DE MS has a DL of approximately 10^?8 M, LC‐MS/MS is ～10^?10 M and saturating Cy5 dye method is ～10^?13 M. Archakov et al. report on an atomic force microscope technique that can yield a DL of 10^?16 M when the target is irreversibly fixed to the bait to avoid the losses due to dissociation kinetics. At that level, over 1 000 000 different proteins can be seen in human plasma. How many biomarkers do you want? Math warning: more equations than figures. Archakov, A. et al., Proteomics 2009, 9, 1326‐1343. Unexplored territory: a catfish pathogen's proteome As genomic and proteomic tools become more powerful and cheaper per base or peptide, we can expect to see more papers like this one by Dumpala et al., focused on an organism of modest economic value. Each paper will, however, contribute a new niche with alternative adaptations for survival. In this case, we are introduced to Edwardsiella ictaluri, a Gram negative pathogen of farm‐raised channel catfish. Enteric septicemia of catfish is the most frequent disease of the commercially farmed catfish and appears in acute and chronic forms. For the work reported here, the bacteria were grown in culture, washed, lysed and separated by 2‐DE TOF/TOF or 2‐D LC‐MS/MS for peptide identification. The combined methods identified 788 unique proteins, including 73 ribosomal proteins, several protein synthesis factors, tRNA synthases and a number of other proteins that could be assigned by orthology to Escherichia coli or Edwardsiella tarda. Dumpala, P. R. et al., Proteomics 2009, 9, 1353‐1363.     

In this issue: Proteomics 5/2009

In this issue of Proteomics you will find the following highlighted articles: Heart (pump) broken? Hearts are pumps within pumps within channels and pumps. Calcium is pumped, potassium, sodium, amino acids, and electrons are all pumped, channeled or driven until, finally, blood is pumped. Failure of one or more pumps leads to a heart attack. This report from Zlatkovic et al. looks at the sub‐proteome associated with hypertensive failure of the K⁺ATP channel and associated cardiomyopathy that develops in KIR6.2 knock‐out mice. Out of >900 reproducible 2‐DE spots, 81 displayed significant over‐ or under‐expression, a number of which validated previously proposed interactions with the Kir6.2 channel. Two‐thirds were down‐regulations, including creatine kinase, adenylate kinase, and lactate dehydrogenase. A total of 114 proteins were ontologically mapped into the K⁺ATP‐dependent sub‐proteome and a role in hypertensive heart failure. Interaction mapping found >240 nodes and >1200 interactions/edges. A good foundation for future work. Zlatkovic, J. et al., Proteomics 2009, 9, 1314‐1325. The deeper you dig, the more you find A classical biochemist interested in protein‐protein interactions purifies his protein away from other proteins, seeking the highest “‐fold purification”. A proteomicist, on the other hand, looks for “consistent contamination” – i.e. association – of the protein of interest with other proteins. This requires high resolution separations and high accuracy concentration determinations. You can only work with species with concentrations above the detection limit (DL) for the detection method. 2‐DE MS has a DL of approximately 10^?8 M, LC‐MS/MS is ～10^?10 M and saturating Cy5 dye method is ～10^?13 M. Archakov et al. report on an atomic force microscope technique that can yield a DL of 10^?16 M when the target is irreversibly fixed to the bait to avoid the losses due to dissociation kinetics. At that level, over 1 000 000 different proteins can be seen in human plasma. How many biomarkers do you want? Math warning: more equations than figures. Archakov, A. et al., Proteomics 2009, 9, 1326‐1343. Unexplored territory: a catfish pathogen's proteome As genomic and proteomic tools become more powerful and cheaper per base or peptide, we can expect to see more papers like this one by Dumpala et al., focused on an organism of modest economic value. Each paper will, however, contribute a new niche with alternative adaptations for survival. In this case, we are introduced to Edwardsiella ictaluri, a Gram negative pathogen of farm‐raised channel catfish. Enteric septicemia of catfish is the most frequent disease of the commercially farmed catfish and appears in acute and chronic forms. For the work reported here, the bacteria were grown in culture, washed, lysed and separated by 2‐DE TOF/TOF or 2‐D LC‐MS/MS for peptide identification. The combined methods identified 788 unique proteins, including 73 ribosomal proteins, several protein synthesis factors, tRNA synthases and a number of other proteins that could be assigned by orthology to Escherichia coli or Edwardsiella tarda. Dumpala, P. R. et al., Proteomics 2009, 9, 1353‐1363.     

Analysis of Human Plasma Proteome by 2DE‐ and 2D nanoLC‐Based Mass Spectrometry

Abstract

We compared the 2DE coupled to MALDI‐TOF‐MS and ESI‐MS/MS analysis (2DE‐MS) and the on‐line 2D nanoLC, followed by nanoESI‐MS/MS analysis (2DLC‐MS), for the separation and identification of proteins in high abundance protein‐depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE‐MS method is amenable to protein spot‐based quantitation, whereas the 2DLC‐MS method may provide an advantage of the high throughput application.     

Protein extraction from the earthworm Eisenia fetida for 2‐DE

We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2‐DE. Sample preparation is a critical step in a 2‐DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2‐DE. The methods generated remarkably different 2‐DE protein spot patterns. We conclude that trichloroacetic acid (TCA)‐A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA‐A gives good distinction, more bands in 1‐DE gels, and the most number of protein spots in 2‐DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2‐DE gels, analyzed them using MALDI‐TOF/TOF MS, and tentatively identified them. The classic TCA‐A method proved to be most useful as a standard method of extracting proteins from E. fetida.     

Proteomic analysis of the fish pathogen <Emphasis Type="Italic">Flavobacterium columnare</Emphasis>

Background  

Flavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.     

Towards functional proteomics of minority component of honeybee royal jelly: The effect of post‐translational modifications on the antimicrobial activity of apalbumin2

This study illustrates multifunctionality of proteins of honeybee royal jelly (RJ) and how their neofunctionalization result from various PTMs of maternal proteins. Major proteins of RJ, designated as apalbumins belong to a protein family consisting of nine members with M_r of 49–87 kDa and they are accompanied by high number of minority homologs derived from maternal apalbumins. In spite of many data on diversity of apalbumins, the molecular study of their individual minority homologous is still missing. This work is a contribution to functional proteomics of second most abundant protein of RJ apalbumin2 (M_r 52.7 kDa). We have purified a minority protein from RJ; named as apalbumin2a, differ from apalbumin2 in M_r (48.6 kDa), in N‐terminal amino acids sequences – ENSPRN and in N‐linked glycans. Characterization of apalbumin2a by LC‐MALDI TOF/TOF MS revealed that it is a minority homolog of the major basic royal jelly protein, apalbumin2, carrying two fully occupied N‐glycosylation sites, one with high‐mannose structure, HexNAc2Hex9, and another carrying complex type antennary structures, HexNAc4Hex3 and HexNAc5Hex4. We have found that apalbumin2a inhibit growth of Paenibacillus larvae. The obtained data call attention to functional plasticity of RJ proteins with potential impact on functional proteomics in medicine.     

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2‐DE. The differential 2‐DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH‐dependent N10 genes using Western blot and real‐time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH‐dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell‐shape mode of pH‐dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH‐responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic‐adaptive mechanism.     

Proteome profile of the developing maize (Zea mays L.) rachis

In this study, we performed the first high‐throughput proteomic analysis of developing rachis (cob) from maize genotype Mp313E. Using two proteomic approaches, 2‐DE and 2‐D LC, we identified 967 proteins. A 2‐D proteome reference map was established. Functional classification of identified proteins revealed that proteins involved in various cellular metabolisms, response to stimulus and transport, were the most abundant.     

Proteomics of rice and Cochliobolus miyabeanus fungal interaction: Insight into proteins at intracellular and extracellular spaces

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice–C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG‐fractionation of total proteins coupled with MS (MALDI‐TOF/TOF and nESI‐LC‐MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS‐PAGE in combination with nESI‐LC‐MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice–C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.     

A comprehensive proteome map of bovine cerebrospinal fluid

Cerebrospinal fluid (CSF) is considered as the most promising body fluid target for the discovery of biomarkers for early diagnosis of neurodegenerative diseases such as Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy in cattle. For the recognition of disease‐associated changes in bovine CSF protein patterns, a detailed knowledge of this proteome is a prerequisite. The absence of a high‐resolution CSF proteome map prompted us to determine all bovine CSF protein spots that can be visualised on 2‐D protein gels. Using state‐of‐the‐art 2‐DE technology for proteome mapping of bovine ante mortem CSF combined with sensitive fluorescent protein staining and MALDI‐TOF/TOF MS for protein identification, a highly detailed 2‐DE map of the bovine CSF proteome was established. Besides the proteins mapped by earlier studies, this map contains 66 different proteins, including 58 which were not annotated in bovine 2‐DE CSF maps before.     

Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation,bottom‐up and top‐down mass spectrometry

Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed.     

Two‐dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brain

We describe a 2‐DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6–11 IPG strips using paper‐bridge loading and on 12% SDS‐PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in‐house proteomics data analysis platform, Proline. The 2‐DE reference map is available via the UCD 2‐DE Proteome Database ( http://proteomics‐portal.ucd.ie:8082 ) and can also be accessed via the WORLD‐2DPAGE Portal ( http://www.expasy.ch/world‐2dpage/ ). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018–10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2‐DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia.