Unintended molecular interactions in transgenic plants expressing clinically useful proteins: The case of bovine aprotinin traveling the potato leaf cell secretory pathway

We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On‐chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast. No visible phenotypic effects of aprotinin were observed for the transgenic lines, but aprotinin retention in the ER was associated with a significant decrease of leaf soluble protein content. A 2‐D gel assessment of control and transgenic lines revealed a possible link between this altered protein content and the down‐regulation of proteins implicated in protein synthesis and maturation. These observations, supported by complementary 2‐DE analyses with potato lines targeting aprotinin to the apoplast, suggest an aprotinin‐mediated feedback in planta negatively altering protein anabolism. From a practical viewpoint, these data illustrate the importance of taking into account not only the characteristics of recombinant proteins expressed in heterologous environments, but also their possible effects on protein accumulation in the host plant factory.     

Targeted serum glycoproteomics for the discovery of lung cancer‐associated glycosylation disorders using lectin‐coupled ProteinChip arrays

To screen for glycoproteins showing aberrant sialylation patterns in sera of cancer patients and apply such information for biomarker identification, we performed SELDI‐TOF MS analysis coupled with lectin‐coupled ProteinChip arrays (Jacalin or SNA) using sera obtained from lung cancer patients and control individuals. Our approach consisted of three processes (i) removal of 14 abundant proteins in serum, (ii) enrichment of glycoproteins with lectin‐coupled ProteinChip arrays, and (iii) SELDI‐TOF MS analysis with acidic glycoprotein‐compatible matrix. We identified 41 protein peaks showing significant differences (p<0.05) in the peak levels between the cancer and control groups using the Jacalin‐ and SNA‐ProteinChips. Among them, we identified loss of Neu5Ac (α2,6) Gal/GalNAc structure in apolipoprotein C‐III (apoC‐III) in cancer patients through subsequent MALDI‐QIT‐TOF MS/MS. Furthermore, subsequent validation experiments using an additional set of 60 lung adenocarcinoma patients and 30 normal controls demonstrated that there is a higher frequency of serum apoC‐III with loss of α2,6‐linkage Neu5Ac residues in lung cancer patients compared to controls. Our results have demonstrated that lectin‐coupled ProteinChip technology allows the high‐throughput and specific recognition of cancer‐associated aberrant glycosylations, and implied a possibility of its applicability to studies on other diseases.     

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.     

Protein reference mapping of dihydrofolate reductase‐deficient CHO DG44 cell lines using 2‐dimensional electrophoresis

Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase‐deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well‐characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2‐DE with various immobilized pH gradients (pHs 3–10, 5–8, and 3–6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver‐stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI‐TOF‐MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.     

Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.     

Product and contaminant measurement in bioprocess development by SELDI‐MS

Bioprocesses for therapeutic protein production typically require significant resources to be invested in their development. Underlying these efforts are analytical methods, which must be fit for the purpose of monitoring product and contaminants in the process. It is highly desirable, especially in early‐phase development when material and established analytical methods are limiting, to be able to determine what happens to the product and impurities at each process step with small sample volumes in a rapid and readily performed manner. This study evaluates the utility of surface‐enhanced laser desorption ionization mass spectroscopy (SELDI‐MS), known for its rapid analysis and minimal sample volumes, as an analytical process development tool. In‐process samples from an E. coli process for apolipoprotein A‐IM (ApoA‐IM) manufacture were used along with traditional analytical methods such as HPLC to check the SELDI‐MS results. ApoA‐IM is a naturally occurring variant of ApoA‐I that appears to confer protection against cardiovascular disease to those that carry the mutated gene. The results show that, unlike many other analytical methods, SELDI‐MS can handle early process samples that contain complex mixtures of biological molecules with limited sample pretreatment and thereby provide meaningful process‐relevant information. At present, this technique seems most suited to early‐phase development particularly when methods for traditional analytical approaches are still being established. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

N‐terminal processing of affinity‐tagged recombinant proteins purified by IMAC procedures

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S‐transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine‐containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7‐triazacyclononane (tacn). The use of this tag‐tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli‐expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP‐1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI‐TOF MS analysis of the cleaved products from the DAP‐1 digestion of the recombinant N‐terminally tagged proteins confirmed the complete removal of the tag within 4‐12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn‐based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli‐expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of differentially accumulating pistil proteins associated with self‐incompatibility of non‐heading Chinese cabbage

Non‐heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), an important vegetable crop in China, exhibits a typical sporophytic self‐incompatibility (SI) system. To better understand the mechanism of SI response and identify potential candidate proteins involved in the SI system of this vegetable crop, the proteomic approach was taken to identify differential accumulating pistil proteins. Pistils were collected at 0 h and 2 h after self‐pollination at anthesis in self‐incompatible and compatible lines of non‐heading Chinese cabbage, and total proteins were extracted and separated by two‐dimensional gel electrophoresis (2‐DE). A total of 25 protein spots that displayed differential abundance were identified by matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry (MALDI–TOF/TOF MS) and peptide mass fingerprinting (PMF). Among them, 22 protein spots were confidently established. The mRNA levels of the corresponding genes were detected by quantitative RT‐PCR. The 22 identified protein spots are involved in energy metabolism (four), protein biosynthesis (three), photosynthesis (six), stress response and defence (five), and protein degradation (four). Among these potential candidate proteins, UDP‐sugar pyrophosphorylase could be involved in sucrose degradation to influence pollen germination and growth. Glutathione S–transferases could be involved in pollen maturation, and affect pollen fertility. Senescence‐associated cysteine protease, which is related to programmed cell death, could be mainly related to self pollen recognition of non‐heading Chinese cabbage. The study will contribute to further investigations of molecular mechanism of sporophytic SI in Brassicaceae.     

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.     

Rapid identification and characterization of Vibrio species using whole‐cell MALDI‐TOF mass spectrometry

Aims: Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish closely related species. This work aimed at evaluating the use of MALDI‐TOF mass spectrometry for the rapid identification of Vibrio (V.) spp. as an advantageous application to rapidly discriminate the most important Vibrio spp. and distinguish Vibrio spp. from closely related bacterial species like Photobacterium damselae and Grimontia hollisae and other aquatic bacteria like Aeromonas spp. Methods and Results: Starting from sub‐colony amounts of pure cultures grown on agar plates, a very simple sample preparation procedure was established and combined with a rapid and automated measurement protocol that allowed species identification within minutes. Closely related species like Vibrio alginolyticus and Vibrio parahaemolyticus or Vibrio cholerae and Vibrio mimicus could thus be differentiated by defining signatures of species‐identifying biomarker ions (SIBIs). As a reference method for species designation and for determination of relationships between strains with molecular markers, partial rpoB gene sequencing was applied. Conclusions: The MALDI‐TOF MS‐based method as well as the rpoB sequence‐based approach for Vibrio identification described in this study produced comparable classification results. The construction of phylogenetic trees from MALDI‐TOF MS and rpoB sequences revealed a very good congruence of both methods. Significance and Impact of the Study: Our results suggest that whole‐cell MALDI‐TOF MS‐based proteometric characterization represents a powerful tool for rapid and accurate classification and identification of Vibrio spp. and related species.     

Study of rat hypothalamic proteome by HPLC/ESI ion trap and HPLC/ESI‐Q‐TOF MS

The proteomic profile of hypothalamus, a key organ of CNS, is explored here by employing two widely used MS techniques, i.e. HPLC/ESI‐ion trap and HPLC/ESI‐quadrupole‐TOF MS. Strong cation exchange is used for the fractionation of peptides and protein search engine MASCOT is employed for data query. One hundred and thirty six proteins with 10 973 peptides were identified by HPLC/ESI‐ion trap MS, while 140 proteins with 32 183 peptides were characterized by HPLC/ESI‐quadrupole‐TOF MS. Among the total 198 proteins identified in both experiments, 78 proteins were common in both sets of conditions. The rest of the 120 proteins were identified distinctly in both MS strategies, i.e. 58 unique proteins were found using the quadrupole‐TOF while 62 were found with the HPLC/ESI‐ion trap. Moreover, these proteins were classified into groups based on their functions performed in the body. Results presented here identified some important signal and cellular defense proteins inevitable for survival in stressed conditions. Additionally, it is also shown that any single MS strategy is not reliable for good results due to loss of data depending on sensitivity of the instrument used.     

Rapid label‐free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome

Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane‐embedded sub‐proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re‐annotation of the theoretical E. coli IMP regarding the sub‐cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC‐MS/MS analysis, we experimentally identified ～45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label‐free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over‐synthesizing the membrane‐embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.     

Life‐style changes of a halophilic archaeon analyzed by quantitative proteomics

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope‐coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N‐termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS‐PAGE, in‐gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI‐TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of ¹²C and ¹³C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.     

Optimization of a phenol extraction‐based protein preparation method amenable to downstream 2DE and MALDI‐MS based analysis of bacterial proteomes

2DE is one of the most efficient and widely used methods for resolving complex protein mixtures. For efficient analysis of complex samples, high‐resolution separation of proteins on 2D gel is essential, and for that purpose good sample preparation is crucial. In this study, we have improvized a method for preparing bacterial total cellular proteome, from a strategy applied earlier to recalcitrant plant tissues, which gave high‐quality resolution on 2DE. The method involving phenol extraction followed by methanol/ammonium acetate precipitation was first optimized for the chemolithotrophic proteobacteria Tetrathiobacter kashmirensis WT001 and Pseudaminobacter salicylatoxidans KCT001 that did not yield quality protein preps in conventional trichloroacetic acid/acetone precipitation method. Subsequently, to validate its general applicability, the method was evaluated against the trichloroacetic acid/acetone precipitation method for two other model bacteria, i.e. Escherichia coli DH5α and Mycobacterium smegmatis mc²6. Identification of at least four proteins each from the outer membrane, periplasm, and cytoplasm of T. kashmirensis by MALDI‐MS not only proved the efficiency of the method in extracting proteins from the different cellular compartments but also the amenability of the obtained protein spots toward MALDI‐MS based identification.