Immobilized pH Gradient Strips Research Articles

English

This study is about proteomic analysis of oral verrucous carcinoma (OVC). The total proteins obtained from tumour and adjacent normal oral mucosa of patients with OVC and oral squamous cell carcinoma (OSCC) were separated with two dimensional electrophoresis (2-DE) by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysed by image master 2D elite. Twenty distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the tryptic digested peptides mixture was performed by using MALDI-TOF-MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were applied to National Center for Biotechnology Information (NCBI), SWISS-PROT and MSDB databases using Mascot software. Then the 2-DE gel imaging showed that 74, 36 and 31 differential protein spots were found between OVC and OSCC, OVC and adjacent normal oral mucosa of OVC (OVCN), OSCC and adjacent normal oral mucosa of OSCC (OSCCN) samples, respectively. By identification of protein spots from 2-DE gels, 20 PMF maps were obtained by MALDI-TOF-MS including recoverin (cancer associated retinopathy (CAR) protein) tumor protein D53 (hD53), zinc finger protein 77 (ZNFpT1) and so on and these protein may play a key role in the carcinogenesis of OVC and OSCC.             Key words: Oral verrucous carcinoma, oral squamous cell carcinoma, two-dimensional electrophoresis, peptide mass fingerprints, matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Proteomic Profiling of Adipose Tissue from Zmpste24−/− Mice, a Model of Lipodystrophy and Premature Aging, Reveals Major Changes in Mitochondrial Function and Vimentin Processing

Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste24, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste24 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste24(-/-) mice as experimental model. We show that Zmpste24 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste24-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste24(-/-) mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste24 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or 2-arachidonoylglycerol, to be dysregulated in Zmpste24(-/-) serum. Together, our findings in Zmpste24(-/-) mice may be useful to unveil the mechanisms underlying adipose tissue dysfunction and its overall contribution to body homeostasis in progeria and other lipodystrophy syndromes as well as to develop novel strategies to prevent or ameliorate these diseases.

Time‐varying migration process of moving neutralization boundary on the immobilized pH gradient strip in the weak‐base rehydration buffer

This paper quantificationally probes into time-varying migration processes of moving neutralization boundary (MNB) on immobilized pH gradient (IPG) strip in ammonia-rehydration buffers. The time-varying migration processes are determined by both time-varying dissociation equilibria of ammonia and position-varying pH environments formed by immobilized carrier ampholytes (CAs) on the IPG strip. Thus, the local dissociation equilibria of ammonia and the position-varying pH are introduced into the recursion equation of position of MNB migrations. The theoretical position-time curves and the velocity-time curves of MNB migrations obtained by the recursion approach were satisfactorily validated by a series of images of boundary migrations from the IPG-MNB experiments by using rehydration buffers with different ammonia concentrations on pH 3-6 IPG strips. The results achieved herein have significant evidence to a quantificational understanding of the mechanism of MNB and IEF.

Identification of Outer Membrane Proteins from an Antarctic Bacterium Pseudomonas syringae Lz4W

Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.

Prefractionation enhances loading capacity and identification of basic proteins from human breast cancer tissues

Many basic proteins (p I > 7) and putative disease biomarkers are not identified using conventional proteomic methods. This study applied a new method to improve the identification of such proteins. Prefractionated basic proteins were compared with total tissue lysates from human ductal carcinoma in situ tissue loaded on basic immobilized pH gradient strips prior to two-dimensional gel electrophoresis (2-DE). Extraction of alkaline proteins was achieved in less than 20 min using a chromatofocusing resin and two buffers in a microcentrifuge tube. Prefractionation showed improved resolution and visualization of low-abundance proteins on 2-DE gels, allowing proteins to be excised, accumulated, trypsin-digested, and identified by liquid chromatography–tandem mass spectrometry. Proteins identified in the prefractionated samples had a higher number of peptides and three times the number of unique basic proteins when compared with total lysates. Low-molecular-weight (LMW, <26 kDa) unique alkaline proteins comprise 75% of those identified in prefractionated samples compared with 25% identified in total lysates, representing a 9-fold increase of LMW proteins due to prefractionation. Prefractionation ultimately increases loading capacity of samples onto the 2-DE gel and leads to better resolution, visualization, and identification of proteins with p I values greater than 7.

Identification for immuno-reactive proteins of &lt;I&gt;Vibrio harveyi&lt;/I&gt; by two-dimensional electrophoresis method

Vibrio harveyi is one of the most serious marine pathogen that can infect a number of aquaculture species.It has caused severe losses to aquaculture industries worldwide.Attempts to control the infection are hampered by lack of effective vaccines and rapid diagnostic kits,the formulation of which could be facilitated by the identification of immunogenic proteins.In this study,an immunoproteome-based approach was developed to identify candidate antigens of Vibrio harveyi for vaccine development.A 2-DE map has been constructed for Vibrio harveyi,in the pI range of 4.0 to 7.0.Strain HY99 was grown in TSA medium for 18 hours at 28 ℃.Total soluble proteins were extracted using lysis buffer and purified with a 2-D clean-up kit.Protein concentrations were determined by 2-D Quant Kit,and the proteins were separated by 2-DE under immobilized pH gradients(IPG).The 2-DE map was obtained from 3 gels run with 7 cm immobilized pH gradient strips and 12.5% SDS-PAGE gels.The electrophoregrams were obtained by coomassie brilliant blue staining.2-DE gels were scanned with Image Master 2D Platinum analyzed by 300 dpi 2-DE image analysis revealed(429 ± 18) protein spots.Then Vibrio harveyi HY99 anti-sera was analyzed for reactivity by Western-blotting against Vibrio harveyi total soluble proteins separated by 2-DE.The 3 maps analyzed revealed 45 pair protein spots by ImageMaster 2D Platinum.15 spots are non-specific-immunoreactive proteins of Vibrio harveyi,and 30 spots are specific-immunoreactive proteins of Vibrio harveyi.These 30 spots were chosen for mass spectrometry identification,and 29 spots were successfully matched with the proteins of NCBInr database(http://www.matrixscience.com).Two isoforms of formate acetyltransferase were proposed.The 30 spots from the 2-DE map corresponded to 28 proteins.None of these identified proteins have previously been reported as immunogenic in Vibrio harveyi.6 proteins are known from other bacterial immunoproteomic analyses.They may be considered to be cross-reactive antigens from other bacterial infections.OmpN were identified a number of times during the immunoproteome analysis of other bacteria,such as Shigella flexneri,Pasteurella multocida,Escherichia coli.OmpW is one of the major outer membrane proteins of Vibrio alginolyticus,and it is an immunoprotein in the report.OmpU is an important virulence factor involved in the adherence of Vibrio vulnificus to the host cells.alanine dehydrogenase,Elongation factor Ts(EF-Ts),cysteine synthase were recognized by anti-sera of Staphylococcus epidermidis.To the best of our knowledge,there are no reports about the immunogenicity of the other remaining 18 identified proteins.Their role in immunoreaction is not fully understood.It is suggested that this study may be valuable for the immuno-proteomics research on Vibrio harveyi.These immunoreactive proteins could be novel candidates for vaccine development.Future studies will evaluate the protection of the 28 proteins by a nasal immunization and challenge.

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a beta-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca(2+) sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased beta myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.

Identification ofHelicobacter pyloriStrain 51 Major Outer Membrane Proteins by Quadrupole Time of Flight Mass Spectrometry

As part of an initial inquiry into the function of the outer membrane proteins (OMPs) of Helicobacter pylori Korean strain 51, we have conducted an extensive proteome analysis via quadrupole time of flight (Q-TOF) mass spectrometry (MS). Fifty one OMPs of H. pylori were purified using sarcosine and resolved via two-dimensional electrophoresis with immobilized pH gradient strips. The most abundant proteins were observed in the alkaline pI regions (6.0~11.0) at molecular masses between 10~100 KDa. Here, 15 spots were identified, representing 9 types of genes (KHP0852, KHP0853, KHP1353, KHP1017, KHP0172, KHP0076, KHP0617, KHP1069, KHP0614) from the sarcosin-insoluble fraction of H. pylori 51. These may be employed in the characterization of the OMPs of H. pylori 51, which will help to identify new potential target proteins for vaccine development and drug therapy.

Regulation of Dauer Formation by O-GlcNAcylation in Caenorhabditis elegans

Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.

Microscopic and Protein Expression Analysis of Grain Weight Variation within the Spike of Triticum aestivum

The kernels possess significant grain weight variation in one wheat (Triticum aestivum L) plant because of their different positions within the spike. In order to understand the molecular basis of weight, a proteomic approach, employing twodimensional electrophoresis and matrix-assisted laser desorptionfionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins between two kinds of kernels, the high weight kernel (large kernel) and the low weight kernel (small kernels) with different positions within spikes of one wheat cultivar, Shannong. Microscopic observation showed that the endosperm cells in large kernels enlarged their volume and accumulated storage materials at grain filling stage (17 days after anthesis, DAP), whilst those in small kernels were mainly in cell division with larger vacuoles during this period. Proteins were extracted from the kernels at this time, and resolved using 24-cm immobilized pH gradient strips with a pH 3–10 linear gradient in the first dimension and a 12.0% sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension. About 750 protein spots in each gel were resolved after electrophoresis and 45 proteins were expressed significantly differently between the two kernels. MALDI-TOF MS characterization of the resolved spots in the two samples enabled us to identify 28 proteins whose levels were altered; 19 and 9 proteins were up-regulated in high and low weight kernels, respectively. In particular, proteins beneficial to materials synthesis and transmission increased distinctly in high weight kernels, while in low weight kernels, proteins involved in cell division were increased. The kernels with different position in spike might be at different physiological status, and this might be one of the causes resulting in grain weight differences within one spike.

Mutant Prion Protein Expression Is Associated with an Alteration of the Rab GDP Dissociation Inhibitor α (GDI)/Rab11 Pathway

The prion protein (PrP) is a glycosylphosphatidylinositol-anchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4-7 and 6-11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor alpha (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway.

Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius

BackgroundTwo-dimensional gel electrophoresis (2-DE) is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF), a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).ResultsAn extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 U/ml Deoxyribonuclease I (DNase I), and 0.25 mg/ml Ribonuclease A (RNase A), combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG) strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF). When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found.ConclusionThese results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.

Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts

Artifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction−oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds.

Annexin A2 Is a C-terminal PCSK9-binding Protein That Regulates Endogenous Low Density Lipoprotein Receptor Levels

The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.

A single lysis solution for the analysis of tissue samples by different proteomic technologies

Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a “one-size fits all” solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and tongue. This solution when combined with a specific sample preparation technique – cryostat sectioning of frozen specimens – simplifies tissue sample preparation and solves most of the difficulties associated with the integration of data generated by different proteomic technologies.

In this issue: Proteomics 15/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:An old dog refines new tricksOld dogs are reputed to be slow learners but they can be subtle manipulators, able to induce younger dogs and gullible owners to share the food dish in their favor or choose the path they prefer. Two‐dimensional gel electrophoresis has been around for more than 25 years but “new and improved” versions continue to appear. Ericsson et al. scramble the order of several steps to get more information out of the combination of IPG/IEF and “shotgun” peptide analysis. Developed for studying mechanisms of drug resistance in small cell lung cancer, the modified protocol fractionates sonically disrupted cells into microsomes and soluble fractions before tryptic digestion and iTRAQ labeling for later quantitation. Digested samples were fractionated on narrow range immobilized pH gradient strips from which they were eluted for MALDI TOF or LC‐MS/MS analysis. Detection and identification of transmembrane proteins were dramatically improved.Ericsson, H. et al., Proteomics 2008, 8, 3008–3018.An evanescent view of a lectin micro‐array: through a glass faintlyLectins have the ability to distinguish closely‐related carbohydrate moieties attached (or not) to other molecules such as proteins, peptides, lipids, cells, etc. In some respects, they are much like antibodies, just not quite as specific. Using an array of 45 different lectins, the glycan portion of glycoproteins can be identified by its binding profile. Here, Uchiyama et al. report the improvements they have made to the reproducibility and sensitivity of the system. The binding of rhodamine‐labeled probes was detected in an evanescent field fluorescence‐based instrument that was capable of reaching, 10pM levels without having to wash off unbound probe. Depositing lectin spots with a non‐contact type printer, at the right humidity, and blocking with a non‐proteinaceous material greatly improved sensitivity.Uchiyama, N. et al., Proteomics 2008, 8, 3042–3050.The innate defense: multiplex proteomic probingThe body's first line of defense against pathogen infection is the innate response. In the case of bacterial infection, it is initiated by the sensing of the universal Gram‐positive cell wall lipopolysaccharide (LPS) component by macrophages primarily (but not solely) through the Toll‐like receptor 4 (TLR4). To understand the regulation of the LPS response, Gu et al. developed a multiplex quantitative proteomic analysis procedure to follow the response of TLR4+ and TLR4− cell lines. The method of choice was amino acid‐coded mass tagging (AACT, also referred to as SILAC). It showed high efficiency of labeling (95%) which eliminated interference with quantitation by the unlabeled fraction. Using triplex labeling of lysine (13C, 15N), the authors confirmed that TLR4− cells did show a response to LPS: 25 proteins were up‐regulated in TLR4+ cells, 5 in TLR4− cells. More than 500 proteins could be quantitated.Gu, S. et al., Proteomics 2008, 8, 3061–3070.

Cover Picture: Proteomics 15/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:An old dog refines new tricksOld dogs are reputed to be slow learners but they can be subtle manipulators, able to induce younger dogs and gullible owners to share the food dish in their favor or choose the path they prefer. Two‐dimensional gel electrophoresis has been around for more than 25 years but “new and improved” versions continue to appear. Ericsson et al. scramble the order of several steps to get more information out of the combination of IPG/IEF and “shotgun” peptide analysis. Developed for studying mechanisms of drug resistance in small cell lung cancer, the modified protocol fractionates sonically disrupted cells into microsomes and soluble fractions before tryptic digestion and iTRAQ labeling for later quantitation. Digested samples were fractionated on narrow range immobilized pH gradient strips from which they were eluted for MALDI TOF or LC‐MS/MS analysis. Detection and identification of transmembrane proteins were dramatically improved.Ericsson, H. et al., Proteomics 2008, 8, 3008–3018.An evanescent view of a lectin micro‐array: through a glass faintlyLectins have the ability to distinguish closely‐related carbohydrate moieties attached (or not) to other molecules such as proteins, peptides, lipids, cells, etc. In some respects, they are much like antibodies, just not quite as specific. Using an array of 45 different lectins, the glycan portion of glycoproteins can be identified by its binding profile. Here, Uchiyama et al. report the improvements they have made to the reproducibility and sensitivity of the system. The binding of rhodamine‐labeled probes was detected in an evanescent field fluorescence‐based instrument that was capable of reaching, 10pM levels without having to wash off unbound probe. Depositing lectin spots with a non‐contact type printer, at the right humidity, and blocking with a non‐proteinaceous material greatly improved sensitivity.Uchiyama, N. et al., Proteomics 2008, 8, 3042–3050.The innate defense: multiplex proteomic probingThe body's first line of defense against pathogen infection is the innate response. In the case of bacterial infection, it is initiated by the sensing of the universal Gram‐positive cell wall lipopolysaccharide (LPS) component by macrophages primarily (but not solely) through the Toll‐like receptor 4 (TLR4). To understand the regulation of the LPS response, Gu et al. developed a multiplex quantitative proteomic analysis procedure to follow the response of TLR4+ and TLR4− cell lines. The method of choice was amino acid‐coded mass tagging (AACT, also referred to as SILAC). It showed high efficiency of labeling (95%) which eliminated interference with quantitation by the unlabeled fraction. Using triplex labeling of lysine (13C, 15N), the authors confirmed that TLR4− cells did show a response to LPS: 25 proteins were up‐regulated in TLR4+ cells, 5 in TLR4− cells. More than 500 proteins could be quantitated.Gu, S. et al., Proteomics 2008, 8, 3061–3070.

Identification of changes in Triticum durum L. leaf proteome in response to salt stress by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

In order to understand the molecular basis of salt stress response, a proteomic approach, employing two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins affected by salinity in wheat (Triticum durum 'Ofanto'). Identification of proteins, whose levels were altered, was performed by comparing protein patterns of salt-treated and control plants. A set of control plants was grown without NaCl addition under the same conditions as the salt-treated plants. Proteins were extracted from the leaves of untreated and NaCl-treated plants, and resolved using 24-cm immobilized pH gradient strips with a pH 4-7 linear gradient in the first dimension and a 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension; the gels were stained with Coomassie and image analysis was performed. Quantitative evaluation, statistical analyses and MALDI-TOF MS characterization of the resolved spots in treated and untreated samples enabled us to identify 38 proteins whose levels were altered in response to salt stress. In particular, ten proteins were downregulated and 28 were upregulated. A possible role of these proteins in response to salinity is discussed.

Establishment of sorghum cell suspension culture system for proteomics studies

This study describes the establishment of sorghum cell suspension culture system for use in proteomics studies. Friable sorghum callus was initiated from young shoots under completely dark conditions on MS medium supplemented with 3 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 2.5 mg/L 1-naphthaleneacetic acid (NAA). Additionally, sorghum cell suspension cultures have been initiated from the friable callus masses in liquid medium with the same composition as the callus initiation medium. Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different protein expression profiles. The sorghum TSP proteome, which is highly complex, is best resolved when separated on large format, 18 cm, pH 4 - 7 isoelectric focusing (IEF) immobilised pH gradient (IPG) strips. On the other hand, the sorghum CF proteome (secretome) is less complex with most proteins being resolved on mini format, 7 cm, pH 3 - 10 IPG strips. Furthermore, narrowing down the pH range from 3 - 10 to 4 - 7 for the CF proteome resulted in improved protein spot resolution.