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Imaging serotonin in live cells
incubation medium were recorded. The data from 20 optical Serotonin, a neurotransmitter derived slices at 1.3-um from tryptophan, has been impllcated in intervals were numerous disorders, ranging from used to construct depression and schizophrenia to hyperthree-dimentension and stroke. Most measurement sional images techniques for serotonin and related neuand to calculate rotransmitters are chemical or electrothe volume of the chemical assays that can only probe the observed serotoextracellular region or imaging techniques that do not directly detect the granular con- nin granules. In tent. Watt W. Webb and co-workees at Cor- the images of nell University move into the cell, measur- cells that coning in real time the serotonin distribution in tained serotonin Images of RBL cells obtained with three-photon excitation bright dots live rat basophilic leukemia cells (RBD fluorescence of serotonin. Cells incubated in 250-pM serotonin (left) ZH3) with a laser-scanning microscope peared that were and control cells (right). (Adapted with permission from the American thcit uses 700-nm wavelength lightfroman not seen in the Association for the Advancement of Science.) IR-pulsed laser to excite three-photon fluo- control images rescence of the native UV transition with- The spots disappeared following immuno- that granules larger than 1 um in diameter out changing the cells contained ~ 50 mM serotonin or, on avergenic stimulation providing further evi8 Images of cells that had been cultured dence that the observed fluorescence was age, 10 serotonin molecules per granule. {Sciencce197,275, 530-32) for 6 h with and without serotonin in the due to serotonin Calibrations revealed J1TV
Double depletion of this peak can be done with small proTo tackle this problem, the Florida researchers grew E. coli on a substrate Consider the advantages of determining teins. However, as the number of elements with natural variation in isotopic double depleted in 13C and 15N, and a protein's molecular mass to within isolated the 107-amino acid protein 1 Da—it would be possible to count the mass increases, finding that peak becomes nearly impossible. At around FKBP. The protein is, in fact, "ennumber of disulfide bridges, identify 15 kDa, the protein monoisotopic peak is riched" in 12C and 14N, and the mopost-translational modifications, recognoisotopic peak in the mass spectrum nize variant amino acid sequences, and below detectability. goesfrom0.65% (relative to the more. New MS techniques, most abundant peak) at natural such as electrospray highisotopic abundance to being resolution FT-ICR, have greatly the most abundant peak for the improved accuracy but still fall "double-depleted" protein. The short of the 1-Da goal. A key mass of FKBP is determined limitation is not knowing the as 11780.01, compared with isotopic composition of each 11780.07 based on the commass spectral peak. puted amino acid sequence. Alan G. Marshall and colleagues at Florida State UniThe double-depletion versity have taken a unique method should allow determinaapproach to achieving 1-Da tion of monoisotopic peaks for resolution. They argue that to proteins with masses as large as accurately and unambiguously 60 kDa. Mass spectral sensitivdetermine a protein mass to ity and detection limits should within 1 Da, the spectroscopist also improve accordingly, and it needs to correctly identify the should be easier to isolate the peaks with a "monoisotopic" desired ions for tandem MS Electrospray ionization FT-ICR spectrum of FK506-binding experiments. (J. Am. Chetn. Soc. mass for example, all 12C, 14N, protein with (top) natural-abundance isotopic protein and 1 1997,119,433-34) (bottom) double-depleted protein sample. H 1B0 and 32S. Identification Analytical Chemistry News & Features, March 1, 1997 157 A
