Proteins Label-free

In one set of experiments a titration of compound is performed to assess its potency in vivo. HeLa cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37° in 5% C02. One day prior to labeling, the cells are seeded in 24-well plates at approximately 60,000 cells per well. The next day, cells are washed with warm (37°) PBS and the medium replaced with 250 41 of methionine-free DMEM containing 10% dialyzed serum (Invitrogen). After a 15-min incubation at 37°, different concentrations of compound are added to the cells (which can range from 1 nM to 50 fiM) and the incubation continued for another 45 min. Anisomycin is used as a positive control at a final concentration of 50 /iM. Fifty-five microcuries of 35S-methionine/cysteine [35S-methionine/cysteine express protein labeling mix (1175 Ci/mmol) (Per-kin-Elmer)] is added to each well (220 /(Ci/ml) and the incubation continued for another 15 min. 

Maehashi, K., Katsura, T., Kerman, K., Takamura, Y., Matsumoto, K., and Tamiya, E. (2007) Label-free protein biosensor based on aptamer-modified carbon nanotube field-effect transitors. Anal. Chem. 79, 782-787. 

Muckenschnabel, R. Falchetto, L. M. Mayr, I. Filipuzzi SpeedScreen label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands. Anal. Biochem. 2004, 324, 241-249. 

Fig. 5.14 Principle of label-free ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). Unbound compounds are separated from PL by passage through a restricted-access column. Subsequently, PL is dissociated at low pH, and active ligands L are detected by LC-ESI-MS.

Fig. 5.17 Demonstration of MS-based bioassay functionality using a plant extract. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron), (a) MS analysis of pure extract by direct injection onto restricted-access column 2 in the absence of affinity protein, (b) Analysis of the same natural extract spiked with digoxin using the label-free MS assay method as shown in Fig. 5.15.

Fig. 2. The principles of protein microarray detection strategies, (a) Label-based detection method, (b) label-free based detection methods.

Kyo, M., Usui-Aoki, K. and Koga, H. (2005) Label-free detection of proteins in crude cell lysate with antibody arrays by a surface plas-mon resonance imaging technique. Anal. Chem. 77, 7115-7121. 

The nitrogen In secondary compounds also appears to be readily mobile and metabolizable. When doubly-labeled nicotine ( C - N) was fed to tobacco (j. rustics), carbon was recovered In alkaloids, free amino acids, pigments, free organic acids and free sugars and nitrogen was recovered In proteins, alkaloids, free amino acids and pigments (17). 

If integral membrane proteins are free to diffuse in the membrane, we expect the same to be true of the individual phospholipid molecules that make up the bilayer. To study the dynamics of these motions, phospholipids were labeled with a fluorescent dye that decomposed irreversibly when it was illuminated by a strong laser. When a laser flash was focused to a small spot on the surface of a cell, the labeled phospholipids in this region abruptly ceased fluorescing (fig. 17.16). Fluorescence then rapidly reappeared as the bleached molecules diffused out of the illuminated region and fresh phospholipids diffused in from outside. 

Fig. 5.3. Time-resolved Raman imaging of cytochrome c, protein, and lipid molecule distributions in a label-free HeLa cell during cytokinesis. The images were taken at 5-min intervals (frame rate of 185s/image). Cytokinesis seen as the change in the distribution of proteins and a high concentration of cytochrome c is observed at positions near the contracting ring. The field of view is 161 x 48 pixels. The pixel size is not specified (reprinted with permission from [29])...

T. D. Gibson, Label-free and reversible iminunosensor based upon an ac impedance interrogation protocol, Anal. Chim. Acta, 537 (2005) 163-168. P. Sarkar, One-step separation-free amperometric biosensor for the detection of protein, Microchem. J., 64 (2000) 283-290. 

K. Kerman, Y. Morita, Y. Takamura and E. Tamiya, Escherichia coli single-strand binding protein-DNA interactions on carbon nanotube-modified electrodes form a label-free electrochemical hybridization sensor, Anal. Bioanal. Chem., 381 (2005) 1114-1121. 

Label-free detection of ligand-aptamer interaction was also demonstrated by means of impedance spectroscopy technique [52,53]. Simultaneously, Radi et al. [52] and Rodriguez et al. [53] reported application of Faradaic impedance spectroscopy (FIS) in detection of interaction of proteins with DNA aptamers. The detection method is based on the measurement of resistance in presence of redox mediator Fe(CN)6-In absence of target protein, the negatively charged aptamer repulse the redox mediator molecules from the sensor surface. In a paper by... 

Another label-free optical detection method—FTIR-ATR—has been applied for detection of thrombin by means of DNA aptamers [73], The antithrombin DNA aptamer previously developed by Tasset et al. [17] was immobilized covalently onto Si surface using UV irradiation method. As a quantitative measure, the area of N-H and CH2 bands was used. This method allowed to detect thrombin with a sensitivity around 10 nmol/L. The specificity of binding of protein to aptamer was also investigated using DNA with no binding site for thrombin. It has been noted that for effective binding study by FTIR-ATR method, the concentration of protein should be kept lower than 100 nmol/L. 

LABELING AND LABEL-FREE APPROACHES FOR PROTEIN QUANTIFICATION... 

Various quantitative and statistical validation processes have been described, accounting for the fact that SpCs tend to be small numbers and vary due to the partial stochasticity of the process. In the example datasets included in this chapter, the relationship between the SpC and protein abundance obtained experimentally is shown in Figure 2, demonstrating that many proteins in bacterial cells are low in abundance while a small subset are highly abundant. Several studies have compared label-free with labeling methods or assessed its statistical validity (93-95). Overall, SpC alone should not be used as a means for absolute quantification (92,96), but it is quite adequate for... 

---