SPECIFIC PROTEIN OCCURRENCE RELATED WITH PHOTOPERIODIC FLOWER INDUCTION IN THE COTYLEDONS OF Pharbitis nil CV. VIOLET

In our experiment, three groups of seedlings of SDP Pharbitis nil cv. violet were sepa-rately treated with three different photoperiods (1,16 h dark period--SD; 2, continuous illumi-nation--CL; 3, 16 h dark treatment with 10 min white light in the middle of the dark period--NB). By analysing proteins in the cotyledons from three groups with 2-D PAGE, we found nodifference in protein pattern between the three groups at 0 or 8 h after photoperiodic treatments.At 24 h after the treatments, a specific protein(MW:19 kD; pI: 4.5)appeared only in the cotyledonsof the seedlings which endured SD. This protein disappeared at 72 h after SD. ActinomycinD could inhibit flowering and the specific protein occurrence when applied to cotyledonsprior to SD, but it had no inhibition effect on flowering as well as the specific proteinoccurrence when applied to cotyledons after SD. Chloroamphenicol, a protein synthesisinhibitor, inhibited flowering when applied to cotyledons prior to or immediately after SD,but it did not i     

High-pressure treatment with silver carp (Hypophthalmichthys molitrix) protein and its allergic analysis

The allergenicity and structural changes of silver carp allergens influenced by high-pressure treatment were studied. We treated the allergens at 100, 200 and 300 MPa for 10, 30 and 60 min at 20° C, used SDS–PAGE to separate the proteins and recognized the allergens by western blotting. Circular dichroism analysis was performed to characterize the structural change. From our study, we can determine that high-pressure treatment did not change the subunit composition, molecular weight or the allergenicity of silver carp allergens, but it did change the structure of the allergens.     

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.     

Comparative two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) of human milk to identify dysregulated proteins in breast cancer

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC‐associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one‐dimensional polyacrylamide gel electrophoresis (1D‐PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC‐MS/MS) analysis. The upregulated proteins in BC versus control are alpha‐amylase, gelsolin isoform a precursor, alpha‐2‐glycoprotein 1 zinc isoform CRA_b partial, apoptosis‐inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860.     

A nonradioactive method for detecting phosphates and polyphosphates separated by PAGE

Nonradioactive polyphosphate (poly(P); (PO(3) (-))(n)) species resolved by PAGE can be detected by hydrolytic degradation of the polyphosphates into orthophosphates (P(i)) with a 5 M HCl solution saturated with NaCl, followed by staining the P(i) degradation products in a 1 M HCl solution of 0.25% w/v methyl green and 1% w/v ammonium molybdate. This method detects down to 0.5 nmol of phosphate as P(i), linear poly(P) (condensed phosphate), pyrophosphate (P(2)O(7) (4) (-)), or cyclic trimetaphosphate ion (P(3)O(9))(3) (-) species. This method improves the current method of staining linear poly(P) longer than four phosphate units with Toludine blue-O after PAGE. This study also shows that Stains-All can visualize resolved linear poly(P) shorter than those visualized by Toluidine blue-O. It is hoped that this sequential hydrolytic degradation and phosphate visualization method for detecting ortho-, linear, and cyclic poly(P) species will be a useful tool, as poly(P) are being discovered in a wide variety of biological systems, and their biochemical roles are still largely unknown.     

Detection of bis(sulfosuccinimidyl) suberate binding in electrophoresis: determination of membrane sidedness of proteins

The applicability of the membrane-impermeant protein cross-linker bis(sulfosuccinimidyl) suberate (BS(3)) to the determination of membrane sidedness of proteins was tested in 3T3-L1 cells and in erythrocytes. Binding of BS(3) to proteins was apparent in electrophoresis. In three proteins of 3T3-L1 cells, protein kinase-Cepsilon, protein kinase-Czeta, and glyceraldehyde-3-phosphate dehydrogenase, BS(3) action was detectable in SDS-PAGE with immunoblotting. This enabled confirmation of the well-known intracellular localization of these proteins. In cathepsin E of erythrocytes, a mobility increase in nondenaturing PAGE was the most prominent effect of BS(3) treatment. A mechanism for the increase in mobility due to BS(3) binding is suggested. Cathepsin E was found to be located at the intracellular side of the membrane, in accordance with existing evidence.     

Simultaneous detection of molecular weight and activity of adenylate kinases after electrophoretic separation

Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark-blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long-wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.     

Loss of PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein

SDS‐PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein‐containing conjugates, mostly pegylated proteins. PEG‐maleimide (MAL) is frequently used to site‐specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C–S linkage between protein and PEG‐MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein was observed. It is a thiol‐independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG‐MAL modified conjugates although PEG‐vinylsulfone conjugates to thiol‐containing proteins also through a C–S linkage. Cleavage kinetics of PEG40k‐MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide‐thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG‐MAL modified protein/peptide by SDS‐PAGE analysis.     

A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis

Study of the complexome - all the protein complexes of the cell - is essential for a better understanding and more global vision of cell function. Using two-dimensional blue native/SDS-PAGE (2-D BN/SDS-PAGE) technology, the cytosolic and membrane protein complexes of Escherichia coli were separated. Then, the different partners of each protein complex were identified by LC-MS/MS. In this report, 306 protein complexes were separated and identified. Among these protein complexes, 50 heteromultimeric and 256 homomultimeric protein complexes were found. Among the 50 heteromultimeric protein complexes, 18 previously described protein complexes validate the technology. In this study, 109 new protein complexes were found, providing insight into the function of previously uncharacterized bacterial proteins.     

Bleach gel: a simple agarose gel for analyzing RNA quality

RNA-based applications requiring high-quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the 'bleach gel' is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality.