An enhanced protein crosslink identification strategy using CID-cleavable chemical crosslinkers and LC/MS(n) analysis

We describe a novel two-step LC/MS(n) strategy to effectively and confidently identify numerous crosslinked peptides from complex mixtures. This method incorporates the use of our gas-phase cleavable crosslinking reagent, disuccinimidyl-succinamyl-aspartyl-proline (SuDP), and a new data-processing algorithm CXLinkS (Cleavable Crosslink Selection), which enables unequivocal crosslink peptide selection and identification on the basis of mass measurement accuracy, high resolving power, and the unique fragmentation pattern of each crosslinked peptide. We demonstrate our approach with well-characterized monomeric and multimeric protein systems with and without database searching restrictions where inter-peptide crosslink identification is increased 8-fold over our previously published data-dependent LC/MS3 method and discuss its applicability to other CID-cleavable crosslinkers and more complex protein systems.     

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also freeze the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.     

Schistosoma mansoni: chemical stabilization of cercariae by aldehydes

Chemically stabilized cercariae of Schistosoma mansoni have been developed by inactivating surface glycoproteins which are essential for their survival. The inactivation was achieved by reaction with 0.01-0.1% glutaraldehyde, 0.1-1% formaldehyde, and 0.37-3.7 microliters citraconic anhydride. The cercariae lost their viability but retained the ability to exclude trypan blue for up to 2 years in a manner similar to live cercariae and in contrast to cercariae killed by other means, which took up the dye immediately. The chemically stabilized cercariae reacted with polyclonal and monoclonal antischistosome antibodies in an indirect immunofluorescence assay for up to 2 years, indicating the retention and preservation of surface antigens. Chemically stabilized cercariae revealed the presence of antischistosome antibodies as early as 1 week after infection when used for immunodiagnosis of mouse and rat infections. The presence of Fc receptors for human IgG on the stabilized cercariae interfered in their use as an immunodiagnostic reagent of human schistosomiasis. The stabilized cercariae were also used to screen cultures for monoclonal antischistosome antibodies. Preliminary results indicated that immunization of mice with glutaraldehyde stabilized cercariae imparted protective immunity to mice.     

Enzyme stabilization towards thermal,chemical and proteolytic deactivation

An enzyme stabilization technique which consists of entrapping protein within a polymeric network has been discussed. The high macromolecular concentration levels which lead to formation of the network are produced as a consequence of polarization phenomena which take place within an unstirred ultrafiltration membrane reactor. Increases in enzyme half-life were generally produced in connection with simple and complex deactivation phenomena of widely different natures (thermal, chemical and proteolytic). Experimental tests have been carried out on the following enzymes: β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21), β-d-fructofuranosidase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23).     

Extracellular ascorbate stabilization: Enzymatic or chemical process?

Ascorbate is stabilized in the presence of HL-60 cells. This stabilization has been questioned as a simple chemical effect. Further properties and controls about the enzymatic nature of this stabilization are described and discussed. Our results showed that cAMP derivatives and cAMP-increasing agents stimulated the ability of HL-60 cells to stabilize ascorbate. On the other hand, tunicamycin, a glycosylation-interfering agent, inhibited this ability. These data, together with hormonal regulation, support the hypothesis of an enzymatic redox system located at the plasma membrane as being responsible for the extracellular ascorbate stabilization by HL-60 cells.     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N, N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1 M solute were heated for 10 min at temperatures between 35-75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1 M stabilizer solutions were frozen at -30°C and freeze-dried for 24 h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N,?N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1?M solute were heated for 10?min at temperatures between 35–75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1?M stabilizer solutions were frozen at ?30°C and freeze-dried for 24?h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

Review physical and chemical aspects of stabilization of compounds in silk

The challenge of stabilization of small molecules and proteins has received considerable interest. The biological activity of small molecules can be lost as a consequence of chemical modifications, while protein activity may be lost due to chemical or structural degradation, such as a change in macromolecular conformation or aggregation. In these cases, stabilization is required to preserve therapeutic and bioactivity efficacy and safety. In addition to use in therapeutic applications, strategies to stabilize small molecules and proteins also have applications in industrial processes, diagnostics, and consumer products like food and cosmetics. Traditionally, therapeutic drug formulation efforts have focused on maintaining stability during product preparation and storage. However, with growing interest in the fields of encapsulation, tissue engineering, and controlled release drug delivery systems, new stabilization challenges are being addressed; the compounds or protein of interest must be stabilized during: (1) fabrication of the protein or small molecule-loaded carrier, (2) device storage, and (3) for the duration of intended release needs in vitro or in vivo. We review common mechanisms of compound degradation for small molecules and proteins during biomaterial preparation (including tissue engineering scaffolds and drug delivery systems), storage, and in vivo implantation. We also review the physical and chemical aspects of polymer-based stabilization approaches, with a particular focus on the stabilizing properties of silk fibroin biomaterials.     

Study of the mitochondria movement using videomicroscopy

A stable cell line CV-1 was obtained for vital observation of the transport of mitochondria in animals cells, which express a fragment of the resident protein of mitochondria marked by yellow fluorescent protein. The parameters and conditions of movement of the mitochondria in living cells were established using fluorescence videomicroscopy. Under the normal conditions, only a small part of mitochondria (ca. 7%) was transported over significant distances, while others were in the state of relative rest. The effective transport of mitochondria strictly depended on the dynamic properties of microtubules. Incubation of cell in a serum-free medium suppressed active transport of mitochondria, thus suggesting its dependence on certain, not yet determined environmental factors.     

Further stabilization of earthworm serine protease by chemical modification and immobilization

Earthworm serine protease is more stable and is less affected by organic solvents and detergent than other proteases. However, it is inactivated, probably by autolysis, at 60 degrees C or above under alkaline conditions. Further stabilization was managed by chemical modification of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and phenylglyoxal to protect the activity from the autolytic inactivation. Stabilization was possible also under acidic conditions, in which the stability of the enzyme was rather low, by immobilization with folded sheet mesoporous material. Thus, further stabilization of the enzyme has been achieved by chemical modification or immobilization.     

Actin crosslinkers: repairing the sense of touch

Cells use actin bundles infused with myosin to exert contractile forces on the extracellular environment. This active tension is essential for cellular mechanosensation. Now, the role of actin crosslinkers in stabilizing and repairing the actin bundles is coming into clearer view.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.     

Assay of ATP synthesis using single giant mitochondria

Two forms of EF-1 are present in the high-speed supernatant fraction from wheat embryo homogenate. In embryos from dry seeds EF-1H is 55% of the total amount of EF-1, while after 40 h of germination this form completely disappears. When germination and protein synthesis are accelerated by means of 6-benzyladenine, the rate of conversion of EF-1H is increased. On the other hand, the block of germination and of the evolution of protein synthesis by abscisic acid, block this conversion; the block of water uptake, that stops germination and causes a decrease in protein synthesis, reverses the conversion of EF-1H to EF-1L, increasing EF-1H from 15% to 40% of the total.     

Exercise-induced mitophagy in skeletal muscle occurs in the absence of stabilization of Pink1 on mitochondria

Maintenance of mitochondrial quality is essential for skeletal muscle function and overall health. Exercise training elicits profound adaptations to mitochondria to improve mitochondrial quality in skeletal muscle. We have recently demonstrated that acute exercise promotes removal of damaged/dysfunctional mitochondria via mitophagy in skeletal muscle during recovery through the Ampk-Ulk1 signaling cascade. In this Extra View, we explore whether Pink1 is stabilized on mitochondria following exercise as the signal for mitophagy. We observed no discernable presence of Pink1 in isolated mitochondria from skeletal muscle at any time point following acute exercise, in contrast to clear evidence of stabilization of Pink1 on mitochondria in HeLa cells following treatment with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Taken together, we conclude that Pink1 is not involved in exercise-induced mitophagy in skeletal muscle.