LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Two‐dimensional blue native/SDS‐PAGE is widely applied to investigate native protein–protein interactions, particularly those within membrane multi‐protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we investigated the potential of using LC‐MS/MS as an alternative for SDS‐PAGE in blue native (BN) analysis of protein complexes. By subjecting equal slices from BN gel lanes to label‐free semi‐quantitative LC‐MS/MS, we determined an abundance profile for each protein across the BN gel, and used these profiles to identify potentially interacting proteins by protein correlation profiling. We demonstrate the feasibility of this approach by considering the oxidative phosphorylation complexes I–V in the native human embryonic kidney 293 mitochondrial fraction, showing that the method is capable of detecting both the fully assembled complexes as well as assembly/turnover intermediates of complex I (NADH:ubiquinone oxidoreductase). Using protein correlation profiling with a profile for subunits NDUFS2, 3, 7 and 8 we identified multiple proteins possibly involved in the biogenesis of complex I, including the recently implicated chaperone C6ORF66 and a novel candidate, C3ORF60.     

Comparison of MS/MS methods for protein identification from 2D-PAGE

We have compared the use of a low resolution MALDI-Ion Trap MS/MS and a high-resolution ESI-TOF-MS/MS for the analysis of spots from 2D gels. The main criteria were speed and accuracy of protein identification. The results obtained using the MALDI-MS/MS system are comparable to those from the LC-MS/MS system in terms of accuracy, but less low-level proteins are identified while the time required for the analysis is dramatically reduced.     

Characterization and gene cloning of telomere-binding protein from tobacco BY-2 cells.

In this study, we performed gel mobility shift assays using tobacco BY-2 nuclei extracts to identify the plant telomere-binding proteins (TBP). Although no complexes were detected using C-strand as a probe, a single DNA-protein complex was detected using single-stranded 32P-(TTTAGGG)4 as a probe. In competition experiments, formation of the complex was inhibited only when an ssG-strand telomere repeat was used as a competitor. These results indicate that the observed band reflects a G-strand specific single-stranded telomere-binding protein (NtGTBP1). We purified the binding protein and subsequently used RT-PCR to isolate a gene encoding the protein. The sequence reveals that the protein (NtGTBP1) is a novel TBP from a higher plant, and a search for conserved domains showed that NtGTBP1 contains two RNA recognition motifs (RRMs).     

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.     

Quantitative protein determination for CYP induction via LC-MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.     

Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS

Both organic solvent and surfactant have been used for dissolving membrane proteins for shotgun proteomics. In this work, two methods of protein solubilization, namely using 60% methanol or 1% SDS, to dissolve and analyze the inner membrane fraction of an Escherichia coli K12 cell lysate were compared. A total of 358 proteins (1417 unique peptides) from the methanol-solubilized protein mixture and 299 proteins (892 peptides) from the SDS-solubilized sample-were identified by using trypsin digestion and 2-D LC-ESI MS/MS. It was found that the methanol method detected more hydrophobic peptides, resulting in a greater number of proteins identified, than the SDS method. We found that 159 out of 358 proteins (44%) and 120 out of 299 proteins (40%) detected from the methanol- and SDS-solubilized samples, respectively, are integral membrane proteins. Among the 190 integral membrane proteins 70 were identified exclusively in the methanol-solubilized sample, 89 were identified by both methods, and only 31 proteins were exclusively identified by the SDS method. It is shown that the integral membrane proteins reflected the theoretical proteome for number of transmembrane helices, length, functional class, and topology, indicating there was no bias in the proteins identified.     

Effect of serine/threonine protein kinases and protein phosphatases inhibitors on mitosis progression in a synchronized tobacco BY-2 culture

In order to investigate the role of various serine/threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells, the influence of cyclin(olomoucine) and Ca²⁺/calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine), and a protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin-dependent protein kinases and protein kinase C caused a prophase delay, reduced the mitotic index, and displaced the mitotic peak as compared with control cells. Inhibition of Ca²⁺/calmodulin-dependent protein kinases enhanced the cells entry into prophase and delayed their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances synchronized BY-2 cells entering into all phases of mitosis.     

Cell cycle-dependent accumulation of a kinesin-like protein, KatB/C, in synchronized tobacco BY-2 cells

Immunoblot analysis with antibodies prepared against highly purified recombinant truncated kinesin-like proteins, KatB(5–249) and KatC(207–754), encoded by the katB and katC genes of Arabidopsis thaliana revealed the presence of a kinesin-like polypeptide, termed KatB/C, in cultured tobacco BY-2 cells. The KatB/C polypeptide cosedimented with microtubules in the presence of a nonhydrolyzable ATP analogue and was released from microtubules in the presence of ATP, both of which are characteristics of kinesin proteins. The amount of KatB/C polypeptide in synchronous BY-2 cells increased during M phase of the cell cycle. Microtubule-based structures present in cells at M phase, such as the spindle and phragmoplast, may be the site of action of the KatB/C protein.     

iTRAQ-coupled 2-D LC-MS/MS analysis of membrane protein profile in Escherichia coli incubated with apidaecin IB

Apidaecins are a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. They are predominantly active against the gram-negative bacteria. Previous studies mainly focused on the identification of their internal macromolecular targets, few addressed on the action of apidaecins on the molecules, especially proteins, of bacterial cell membrane. In this study, iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered membrane proteins of Escherichia coli cells incubated with one isoform of apidaecins--apidaecin IB. Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB. Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids. Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.     

Spotlight: Assembly of protein complexes by integrating graph clustering methods

As is generally assumed, clusters in protein–protein interaction (PPI) networks perform specific, crucial functions in biological systems. Various network community detection methods have been developed to exploit PPI networks in order to identify protein complexes and functional modules. Due to the potential role of various regulatory modes in biological networks, a single method may just apply a single graph property and neglect communities highlighted by other network properties.     

iTRAQ-coupled 2-D LC-MS/MS analysis of cytoplasmic protein profile in Escherichia coli incubated with apidaecin IB

Apidaecins refer to a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. Accumulating evidence that proline-rich antimicrobial peptides are not-toxic to human and animal cells makes them potential candidates for the development of novel antibiotic drugs. However, the mechanism of action was not fully understood. In this study, antibacterial mechanism of apidaecins was investigated. iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered cytoplasmic proteins of Escherichia coli incubated with one isoform of apidaecins--apidaecin IB. The production of the chaperonin GroEL and its cofactor GroES, which together form the only essential chaperone system in E. coli cytoplasm under all growth conditions, was decreased in cells incubated with apidaecin IB. The decreasing of the GroEL-GroES chaperone team was further found to be involved in a new antibacterial mechanism of apidaecins. Our findings therefore provide important new insights into the antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.     

Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin

Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G(1) phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q(10). In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.     

Monitoring farnesol-induced toxicity in tobacco BY-2 cells with a fluorescent analog

In a previous study (A. Hemmerlin, T.J. Bach, Plant Physiol. 123 (2000) 1257-1268), we have demonstrated that above a critical concentration, treatment with all-trans-farnesol induces cell-death in Nicotiana tabacum L. cv Bright Yellow-2 (TBY-2) cells. Now we used a fluorescent analog of farnesol (Fol(FLUO)), in which an isoprene unit is replaced by the fluorochrome 7-nitrobenz-2-oxa-1,3-diazol-4-yl, to visualize how cell integrity is affected. Fol(FLUO) exhibited the same toxicity as the natural compound and was shown to be readily taken up by TBY-2 cells, followed by integration into subcellular membrane structures. Although the plasma membrane seemed not to be labeled, Fol(FLUO) was associated with the tonoplast, endoplasmic reticulum, and Golgi apparatus or lipid bodies. Longer exposure times and increased Fol(FLUO) accumulation triggered the formation and proliferation of new membrane structures of as yet unknown function. Finally, at even higher and clearly cytotoxic concentrations of the analog, the cell contents became clearly disorganized, with cell swelling and ultimately plasmolysis.     

Purification and characterization of plant dynamin from tobacco BY-2 cells

We purified an 84 kDa polypeptide from the MAP (microtubule-associated protein) fraction of tobacco BY-2 cultured cells. LC/MS/MS (liquid chromatography-tandem mass spectrometry) analysis revealed that this polypeptide is a tobacco homolog of AtDRP3 (Arabidopsis thaliana dynamin-related protein 3). Electron microscopy revealed that NtDRP3 (Nicotiana tabacum dynamin-related protein 3) assembles to form a filamentous structure. When GDP was added to the NtDRP3 fraction, the filaments disappeared and many particles appeared. Biochemical analysis revealed that NtDRP3 could bind to and bundle both microtubules and actin filaments in vitro.     

Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS

Targeted quantification of proteins is a daily task in biological research but often relies on techniques such as western blotting that are only barely quantitative. Here we present a broadly applicable workflow for protein quantification from unpurified whole-cell extracts that can be completed in less than 3 d. Without prefractionation or affinity enrichment, a whole-cell extract is trypsin-digested in an acetonitrile-containing ammonium carbonate buffer and high-molecular-weight compounds are removed by filtration. A normalization strategy, which involves endogenous reference proteins, facilitates the determination of relative changes in protein expression without requiring isotope labeling or standard addition. On a triple-quadrupole mass spectrometer, we demonstrate standard-free quantification of yeast proteins present over five orders of magnitude and present at ≥500 copies per cell. Liquid chromatography/multiple reaction monitoring (LC-MRM)-based proteomics is therefore a next-generation alternative to western blotting, as it allows simultaneous and reliable quantification of multiple endogenous proteins without the need for enrichment, isotope labeling or use of antibodies.     

Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells

In a functional genomic screen performed by combining an Arabidopsis–yellow fluorescent protein (YFP)-fused complementary DNA (cDNA) library, rat fibroblasts as host and automatic microscopy, we found a short protein with a predictable trans-membrane domain encoded on chromosome 2. In rat fibroblasts, its pattern of distribution was to various organelle-like structures. From the databases, we learned that it has another family member in Arabidopsis and homologs in several other plants, Chlamydomonas and fungi, with a highly conserved N-terminal region. We named this protein from Arabidopsis short membrane protein (SMP) 2. No SMP homologs were found in mammalian sequence databases. When the full-length cDNAs of SMP2 was fused to YFP under the 35S promoter, comparable distribution was observed in Nicotiana benthamiana leaves, suggesting an unknown, evolutionarily conserved localization signal. Similar localization was observed when SMP2 was expressed in N. benthamiana leaves under the control of its own 5′ regulatory sequences. Colocalization studies with green fluorescent protein and red fluorescent protein chimeras revealed its colocalization with chloroplasts, peroxisomes, and mitochondria. No localization of SMP2 was observed in the Golgi. Immunostaining with specific antibodies corroborated the SMP2 localization to the three organelles.     

Cadmium and zinc-mediated oxidative burst in tobacco BY-2 cell suspension cultures

Generation of reactive oxygen species (ROS) constitutes an important first reaction under many stress conditions in plants. We demonstrate that Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures, generate superoxide radical and hydrogen peroxide upon treatment with cadmium and zinc. Addition of catalase and N,N-diethyldithiocarbamate (DDC) decreased the level of H₂O₂, whereas superoxide dismutase (SOD) induced a slight increase of the H₂O₂ production. The effects of catalase, DDC and SOD on the heavy metal-induced ROS production indicate that it occurs outside of the cells, and that at least part of the hydrogen peroxide is produced by dismutation of the superoxide radical (O₂ ^·−). The effect of pretreatment of the cell cultures with commonly used mammalian NADPH oxidase inhibitors was also tested. Strong inhibitions of cadmium and zinc-mediated ROS production were obtained with the flavoprotein inhibitors—diphenylene iodonium (DPI) and quinacrine and with an inhibitor of b-type cytochromes—imidazol. Membrane permeable-N-ethyl maleimide (NEM) and iodoacetate, and membrane non-permeable thiol reagents—para-chloromercuribenzoic acid (pCMBS) also inhibited the ROS production. These results suggested that the enzyme responsible for cadmium and zinc-induced ROS production in tobacco cells contains a flavocytochrome. They also show the importance of intra- and extracellular thiol groups in the observed stress reaction. The induction of ROS production with heavy metals showed properties comparable to the elicitor-induced oxidative burst in other plant cells.     

Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC-MS/MS

In this paper, we describe the use of iTRAQ (isobaric Tags for Relative and Absolute Quantitation) tags for comparison of protein expression levels between multiple samples. These tags label all peptides in a protein digest before labeled samples are pooled, fractionated and analyzed using mass spectrometry (MS). As the tags are isobaric, the intensity of each peak is the sum of the intensity of this peptide from all samples, providing a moderate enhancement in sensitivity. On peptide fragmentation, amino-acid sequence ions also show this summed intensity, providing a sensitivity enhancement. However, the distinct distribution of isotopes in the tags is such that, on further fragmentation, a tag-specific reporter ion is released. The relative intensities of these ions represent the relative amount of peptide in the analytes. Integration of the relative quantification data for the peptides allows relative quantification of the protein. This protocol discusses the rationale behind design, optimization and performance of experiments, comparing protein samples using iTRAQ chemistries combined with strong cation exchange chromatographic fractionation and MS.     

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS

Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer.