Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.     

Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds

A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available-selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins.     

Site-specific chemical labeling of long RNA molecules

Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range of functional groups.     

Microwave‐assisted 18O labeling of Fmoc‐protected amino acids

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of ¹⁸O‐labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave‐assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

In vivo imaging of retrogradely transported synaptic vesicle proteins in Caenorhabditis elegans neurons

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.     

Uniform and Residue-specific 15N-labeling of Proteins on a Highly Deuterated Background

A general method for stable-isotope labeling of large proteins is introduced and applied for studies of the E. coli GroE chaperone proteins by solution NMR. In addition to enabling the residue-specific (15)N-labeling of proteins on a highly deuterated background, it is also an efficient approach for uniform labeling. The method meets the requirements of high-level deuteration, minimal cross-labeling and high protein yield, which are crucial for NMR studies of structures with sizes above 150 kDa. The results obtained with the new protocol are compared to other strategies for protein labeling, and evaluated with regard to the influence of external factors on the resulting isotope labeling patterns. Applications with the GroE system show that these strategies are efficient tools for studies of structure, dynamics and intermolecular interactions in large supramolecular complexes, when combined with TROSY- and CRINEPT-based experimental NMR schemes.     

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.     

Tyramide Signal Amplification Method in Multiple-Label Immunofluorescence Confocal Microscopy

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.     

Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.     

Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ～100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We also show the unique power of our method in single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.     

Improved method for differential expression proteomics using trypsin-catalyzed 18O labeling with a correction for labeling efficiency

Quantitative strategies relying on stable isotope labeling and isotope dilution mass spectrometry have proven to be a very robust alternative to the well established gel-based techniques for the study of the dynamic proteome. Postdigestion 18O labeling is becoming very popular mainly due to the simplicity of the enzyme-catalyzed exchange reaction, the peptide handling and storage procedures, and the flexibility and versatility introduced by decoupling protein digestion from peptide labeling. Despite recent progresses, peptide quantification by postdigestion 18O labeling still involves several computational problems. In this work we analyzed the behavior of large collections of peptides when they were subjected to postdigestion labeling and concluded that this process can be explained by a universal kinetic model. On the basis of this observation, we developed an advanced quantification algorithm for this kind of labeling. Our method fits the entire isotopic envelope to parameters related with the kinetic exchange model, allowing at the same time an accurate calculation of the relative proportion of peptides in the original samples and of the specific labeling efficiency of each one of the peptides. We demonstrated that the new method eliminates artifacts produced by incomplete oxygen exchange in subsets of peptides that have a relatively low labeling efficiency and that may be considered indicative of false protein ratio deviations. Finally using a rigorous statistical analysis based on the calculation of error rates associated with false expression changes, we showed the validity of the method in the practice by detecting significant expression changes, produced by the activation of a model preparation of T cells, with only 5 microg of protein in three proteins among a pool of more than 100. By allowing a full control over potential artifacts, our method may improve automation of the procedures for relative protein quantification using this labeling strategy.     

Use of dinitrophenol-IgG conjugates to detect sparse antigens by immunogold labeling

We describe a novel method for localizing sparse antigens in thin sections by protein A-gold labeling. The primary antibody is applied to fixed and detergent-permeabilized cells. The cells are then incubated with an anti-antibody that has been labeled with multiple dinitrophenol residues. The cells are next fixed again with glutaraldehyde and osmium tetroxide fixatives before embedding in Eponate. When thin sections are prepared, the dinitrophenol residues are readily detected with a tertiary anti-DNP antibody followed by protein A-gold labeling. This method offers good sensitivity along with superior morphology. Our test antigen for this method was the receptor for low-density lipoprotein, an antigen which had evaded detection by protein A-gold using ultra-thin cryosections.     

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

Background

Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.

Methodology

A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.

Conclusions

We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.     

Protein aggregation and degradation during iodine labeling and its consequences for protein adsorption to biomaterials

Protein adsorption on modified and unmodified polymer surfaces investigated through radiolabeling experiments showed a tendency for higher than expected albumin and immunoglobulin G (IgG) adsorption. Possible enhanced protein aggregation and degradation caused by the iodine labeling method used were analyzed through chromatography and spectroscopy techniques. Results show that the iodine labeling method using chloramine-T (CAT) as an oxidizing agent can cause both enhanced aggregation and fragmentation of proteins. Albumin shows an enhanced tendency to aggregate after iodine labeling using the CAT method, and higher amounts of fragmentation are observed for CAT-labeled IgG molecules relative to unlabeled IgG molecules as well as to IgG molecules labeled using the Iodo-Gen method. These results show that the widely applied method of radioisotope labeling for quantitative assessment of protein adsorption should be used with caution and preferably should be validated by a label-free methodology for each combination of radiolabel and protein. The results obtained in this study can be used to optimize investigation of protein adsorption on surfaces of materials for biomedical devices.     

Immunocytochemical determination of the subcellular distribution of ascorbate in plants

Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations.     

An economical method for producing stable-isotope labeled proteins by the <Emphasis Type="Italic">E. coli</Emphasis> cell-free system

Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein using Escherichia coli cell extract-based CF. This method takes advantage of endogenous metabolic conversions to generate SI-labeled asparagine, glutamine, cysteine, and tryptophan, which are much more expensive than the other 16 kinds of SI-labeled amino acids, from inexpensive sources, such as SI-labeled algal amino acid mixture, SI-labeled indole, and sodium sulfide, during the CF reaction. As compared with the conventional method employing 20 kinds of SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost with the newly developed method. Therefore, our method solves the cost problem of the SI labeling of proteins using the CF.     

Optimization and quality assessment of the post-digestion O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry

The post-digestion ¹⁸O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion ¹⁸O labeling, the optimal ¹⁸O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited ¹⁸O incorporation depending on concentration. However, a urea concentration between 1 and 2 M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1–2 M and pH 4.5, 98.4 ± 1.9% for a standard protein mixture and 97.2 ± 6.2% for a complex biological sample. For a 1:1 mixture analysis of the ¹⁶O- and ¹⁸O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05 ± 0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion ¹⁸O labeling method.     

Effect of the Fluorescein to Protein Ratio on Anti-Treponema pallidum Conjugates

The effect of using different labeling techniques and various amounts of fluorescein-isothiocyanate was tested with several pools of antisera to Treponema pallidum. Optimal labeling occurred in all serum pools at a fluorescien to protein ratio of 15. The use of pure dye and the dialysis method of labeling is recommended as the method of choice because of its effectiveness in the reduction of nonspecific staining.