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SERS gets the fat They aren't as glamorous as DNA and proteins, but lipids are an important class of biological compounds not to be overlooked. In most mammals, lipids—typically long-chain fatty acids, their parent triglycerides, and wax esters—are secreted by the sebaceous glands. The in vivo chemistry of these compounds, however, has proven difficult to unravel because of the restricted size of the glands, the mixing of skin electrolytes with lipids to form emulsions, and the presence of compounds with similar structures. Michael D. Morris and colleagues from the University of Michigar attack this problem by testing surfaceenhanced Raman scattering (SERS) as an analysis tool with model sebaceous-gland secretions Silver microprobes on silicon substrates and 75-um silver wire were used as the SERS surfaces. The silicon-substrate microprobes, in particular, have the geometry and demonstrate the mechanical strength and flexibility to be inserted into sebum pores. A 633-nm HeNe laser was used for excitation, and a "T'-junction capillary generated 4 nL/h flows, which simulate glandular secretions. The authors find that fatty acids are strongly adsorbed to the silver microprobe and can be identified in the presence of other lipids. Moreover, the probes are useful for studying the spatial distribution of lipids and their corresponding fatty acids. G4^>/>/. Spectrosc. 1998,52,265-69)
SERS spectra on microprobe alter lipid exposure times of (A) 1 min, (B) 3 min, (C) 5 min; acquisition time of 180 s. (Adapted with permission. Copyright 1998 Society for Applied Spectroscopy.)
An affinity for epitopes One way to characterize cellular structures is to raise antibodies against them and characterize the nature of the antigen recognized. The standard combination of antibody screening methods (western blot analysis, enzymatic treatment, and cDNA library screening) is often of no use if the antibody recognizes nonlinear or discontinuous epitopes or if the target protein is poorly represented in the cDNA library. Simon Gaskell and co-workers at the University of Manchester Institute of Science and Technology and the University of Manchester (both in the U.K.) describe an alternative method for the epitope analysis of monoclonal antibodies that combines immunoaffinity isolation of the antigen with mass spectrometric analysis In this method, the reaction of the antigen and the antibody takes place in solution directly from the cellular ly-
Different neurons, differing peptides iNeurons uiat are funcuonauy related can contain overlapping yet differing sets of peptides. iv. W. Li and co-workers at vnje Universiteit (The Netherlands), Micromass (U.K.), and the University of CaliforniaSan Francisco use MALDI MS to detect peptides in VD1 and RPD2 neurons from the brain of the mollusk Lymnaea stagnalis. These neurons produce peptides that modulate the animal's heartbeat; but MALDI mass spectra reveal that the VD1 neuron contains peptides not found in RPD2. Using a hybrid magnetic-sector-time of flight tandem mass spectrometer, they could identify the complete sequence of one of the unique peptides {Lymnaea small cardioactive peptide [SCP]a) and the partial sequence of another (SCPb) from collision-induced dissociation mass spectra. The researchers used in situ hybridization to independently determine the expression pattern of the SCPs. In situ hybridiza-
sate. They develop the method with a well-understood Tyl retrotransposon from the yeast Saccharomyces cerevisiae. A monoclonal antibody that binds to a known epitope is attached to a magnetic bead and used to fish out the Tyl Gag protein from the solution. The magnetic beads allow the sample to be concentrated with a magnetic bar, avoiding contamination by cellular debris. The sequence of the epitope-containing peptide was confirmed by electrospray MS/ MS. The authors demonstrated that the immunoamnity MS technique can identify a linear epitope that has no posttranslational modifications, and they suggest that the MS analysis could also characterize posttranslationally modified epitopes. However, it remains to be seen if the method will work well for conformational epitopes (in which the participating residues are adjacent in space but not in the primary sequence) (J. Am. Soc. Mass Spectrom. 1998, 9, 208-15)
tion with a SCPa cRNA probe confirmed the presence of the SCP transcript in VD1 but not in RPD2. They could also identify VD1 and RPD2 peptides in MALDI mass spectra of a Lymnaea heart biopsy sample from the auricle and demonstrated that the peptides affected contractions in vitro. {Biochemistry 1998,37,2070-76)
Peptide profiles of (A) single VD1 neuron and (B) single RPD2 neuron.
Analytical Chemistry News & Features, May 1, 1998 3 0 3 A
