Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions

In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.     

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.     

An investigation into the human serum "interactome"

The protein content of human serum is composed of a millieu of proteins from almost every type of cell and tissue within the body. The serum proteome has been shown to contain information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases. Unfortunately, the dynamic range of protein abundance, ranging from > mg/mL level to < pg/mL level, renders complete characterization of this proteome nearly impossible with current analytical methods. To study low-abundance proteins, which have potential value for clinical diagnosis, the high-abundant species, such as immunoglobulins and albumin, are generally eliminated as the first step in many analytical protocols. This step, however, is hypothesized to concomitantly remove proteins/peptides associated with the high-abundant proteins targeted for depletion. In this study, immunoprecipitation was combined with microcapillary reversed-phase liquid chromatography (microRPLC) coupled on-line with tandem mass spectrometry (MS/MS) to investigate the low-molecular-weight proteins/peptides that associate with the most abundant species in serum. By this targeted isolation of select highly abundant serum proteins, the associated proteins/peptides can be enriched and effectively identified by microRPLC-MS/MS. Among the 210 proteins identified, 73% and 67% were not found in previous studies of the low-molecular-weight or whole-serum proteome, respectively.     

An insight into the abundant proteome of 46BR.1G1 fibroblasts deficient of DNA ligase I

This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.     

Shotgun proteomic analysis of microdissected postmortem human pituitary using complementary two-dimensional liquid chromatography coupled with tandem mass spectrometer

The pituitary is responsible for multiple homeostatic functions including metabolism, growth and reproduction. Proteome analysis offers an efficient approach for a comprehensive analysis of pituitary protein expression. The pituitary is usually acquired from postmortem specimens, which may potentially affect the proteome profile by proteolysis. The aim of this study was to determine whether the postmortem pituitary could be used in proteomic analysis combining with Laser capture microdissection (LCM). Digested peptides from LCM captured prolactin (PRL) cells were separated by two dimensional-nanoscale liquid chromatography (2D-nanoLC/MS) and characterized by tandem mass spectrometry (MS). All MS/MS spectrums were searched by SEQUEST and a proteome of 1660 proteins was identified. Category analysis of the proteome revealed an extensive unbiased access to cell component proteins with diverse functional characteristics. The results demonstrated the ability of using 2D-nanoLC/MS to perform sensitive proteomic analysis on limited protein quantities through microdissection. Detailed comparisons between the proteome in question and the one derived from the prolactinoma controls at peptide and protein levels indicated that the two proteomes had similar characters. Overall, our results revealed for the first time the possibility of use of postmortem human pituitary for proteomic research which is important for further studies on disease biomarker identification and molecular mechanisms of prolactinoma tumorigenesis.     

Proteome and phosphoproteome of primary cultured pig urothelial cells

Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer-related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2-D gel electrophoresis and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La(3+) enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non-phosphopeptides. The 2-DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder-related diseases.     

Towards a two-dimensional proteome map of Mycoplasma pneumoniae

A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.     

Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome.     

Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999).

By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research. In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique. We report here the identification of 92 novel protein spots on the yeast 2-D protein map. These identifications extend the number of protein spots identified on our yeast reference map to 401. These spots correspond to the products of 279 different genes. They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry. These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM).     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

A two-dimensional proteome map of Shigella flexneri

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S. flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology. Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program. In total, among the 488 proteins spots processed, 388 proteins were identified. The identified proteins represented 169 genes. By comparing results of Mascot search against databases of Escherichia coli and genomes of S. flexneri 2a, one S. flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences. Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc. Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn.     

A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology. This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago. Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified. In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7. The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control. Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot. In a few selected cases evidence for phosphorylation of these proteins is presented. The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B. subtilis proteome such as alkaline proteins as well as extracellular proteins. A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.     

Proteomics: the move to mixtures.

Proteomics can be defined as the systematic analysis of proteins for their identity, quantity and function. In contrast to a cell's static genome, the proteome is both complex and dynamic. Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). However, this technique is under scrutiny because of a failure to detect low-abundance proteins from the analysis of whole cell lysates. Alternative approaches integrate a diversity of separation technologies and make use of the tremendous peptide separation and sequencing power provided by MS/MS. When liquid chromatography is combined with tandem mass spectrometry (LC/MS/MS) and applied to the direct analysis of mixtures, many of the limitations of 2DE for proteome analysis can be overcome. This tutorial addresses current approaches to identify and characterize large numbers of proteins and measure dynamic changes in protein expression directly from complex protein mixtures (total cell lysates).     

Construction of a comprehensive beer proteome map using sequential filter‐aided sample preparation coupled with liquid chromatography tandem mass spectrometry

The quality traits of beer, which include flavor, texture, foam stability, gushing, and haze formation, rely on contributions from beer proteins and peptides. Large‐scale proteomic analysis of beer is gaining importance, not only with respect to authenticity of raw material in beer but also to improve quality control during beer production. In this work, foam proteins were first isolated from beer by virtue of their high hydrophobicity. Then sequential filter‐aided sample preparation coupled with liquid chromatography and tandem mass spectrometry was used to analyze both beer protein and foam protein. Finally, 4692 proteins were identified as beer proteins, and 3906 proteins were identified as foam proteins. In total, 7113 proteins were identified in the beer sample. Several proteins contributing to beer quality traits, including lipid transfer protein, serpin, hordein, gliadin, and glutenin, were detected in our proteins list. This work constructed a comprehensive beer proteome map that may help to evaluate potential health risks related to beer consumption in celiac patients.     

Proteome analysis in the bovine adrenal medulla using liquid chromatography with tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS)-based proteomics has been used to identify soluble proteins in the bovine adrenal medulla. This gland is a major source of hormones, opioids, neurotransmitters, and several vital proteins. The adrenal medulla proteins were first purified using ammonium sulfate precipitation. The resulting proteins were then pre-fractionated with a C-4 high-performance liquid chromatography (HPLC) column. Each 2-min HPLC fraction was digested with trypsin, and separated further and analyzed using capillary liquid chromatography/tandem mass spectrometry (capLC/nanospray-MS/MS) to map the proteome of the adrenal medulla. The parent mass and sequence ion information thus obtained for tryptic peptides was used to search the NCBInr database using the SEQUEST search engine. A total of 195 proteins were identified, of which 71 had good scores (delta correlation value greater than 0.1, preliminary score above 200, and cross-correlation value above 2.5). The prominent proteins thus identified are secretogranin I precursor, chromogranin A, proenkephalin A precursor, myosin X, hemoglobin beta chain, hemoglobin alpha chain, heat shock protein 10 kDa, and replicase.     

Proteomics of Caenorhabditis elegans over-expressing human alpha-synuclein analyzed by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry: identification of actin and several ribosomal proteins as negative markers at early Parkinson's disease stages

It has been known that the over-expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease (PD), leads to neurodegeneration in PD models. In this study, the changes in protein expression between the transgenic over-expressing human alpha-synuclein wild type (alpha-synWT) and the control Caenorhabditis elegans were elucidated by fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) proteome analysis, which is a highly selective, sensitive, repeatable and quantitative method for protein identification. Because the alpha-synuclein wild-type worms showed moderate levels of dopamine loss without overt behavioral abnormalities, it was suggested that the changes in proteins in the alpha-synWT are related in the sequence of the formation of Lewy bodies. Among more than 400 protein peaks detected, actin and several ribosomal proteins were identified for the first time as negative markers at early PD stages. Actin was suggested to be one of the important targets in the elucidation of the etiology of neuronal diseases such as PD or other synucleinopathies.     

The human heart proteome: Two-dimensional maps using narrow-range immobilised pH gradients

The analysis of complex proteomes is undertaken using a variety of techniques and technologies such as 2-DE, surface-enhanced laser desorption ionisation, and various types of MS. In order to overcome the complexities of protein expression in discrete proteomes, sample fractionation has become an important aspect of proteomic experiments. The use of narrow-range IPGs (nrIPGs) is of special importance using the 2-DE proteomics workflow, since an enhanced visualisation of a given proteome is achieved through an improved physical separation and resolution of proteins. The work described in this paper presents a series of protein maps of the human heart left ventricle proteome that have been generated using nrIPGs for the first, IEF, dimension of 2-DE. A total of 374 gel spots were excised from seven different pH gradients, covering the range pH 3-10, giving rise to a total of 388 identifications from 110 unique proteins. Using Gene Ontologies (GOs), the identified proteins were found to be associated with 97 types of GO Process, 144 types of GO Function, and 54 types of GO Component. It is hoped that the maps presented in this paper will be of use to other researchers for reference purposes.     

Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry

Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.     

Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast

As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins.