news
ANALYTICAL CURRENTS Separating mixtures using fluorous tags
(a) C5H11 Me
Several strategies exist for isolating and characterizing the huge mixtures of compounds typically generated by combinatorial syntheses, but this new approach could be the most efficient yet. Dennis Curran and co-workers at the University of Pittsburgh use fluorinated tags and fluorous chromatography to separate the products of a solution-phase mixture synthesis without purifying the products before the last reaction. The approach was demonstrated by synthesizing various analogues of the natural product mappicine. In the simplest example, the two enantiomeric forms of mappicine were synthesized as a mixture and separated. The first of the four steps in the process was to tag a precursor molecule with a label containing –C6F13 or –C8F17. Then the tagged precursors were pooled, and after the last reaction step, the mixture was run through a column, which separated the compounds by increasing fluorine content. Finally, the purified enantiomers were detagged and the enantiomers isolated. The same 4-step synthesis was taken through a combinatorial-style sequence that resulted in 100 mappicine-based products, which were collected as 25
fluorous-tagged mixtures that contained 4 components. Each 4-component mixture was analyzed by HPLC using the fluorine-separating column and UV and mass spectrometric detection. Although there was no prior purification and an average overall yield of 11%, 99 of the 100 compounds were identified, and the authors say that 87 of these could be isolated in pure form. Even better results are obtained if the products are purified during an intermediate step. The demonstration synthesis used 0.1- to 0.2-g quantities, but larger-scale syntheses are only limited by the size of commercial fluorous HPLC columns. Finally, the authors believe that it is possible to scale up the final HPLC mixture separations from 4-compound pools to as many as 10-compound pools. (Science 2001, 291, 1766–1769)
O N N R1 RfCH2CH2(iPr)2SiO
R1 = Pr, Rf = C4F9 R1 = Et, Rf = C4F13 R1 = iPr, Rf = C8F17 R1 = CH2CH2C6H11, Rf = C10F21 C8F17
Internal standard
0
8.0
(b)
C4F9
16.0
C6F13
24.0
C10F21
32.0
40.0 (min)
Me O N N R1 RfCH2CH2(iPr)2SiO
Internal standard
0
8.0
C4F9
16.0
R1 = Pr, Rf = C4F9 R1 = Et, Rf = C4F13 R1 = iPr, Rf = C8F17 R1 = CH2CH2C6H11, Rf = C10F21
C6F13
24.0
C8F17 C10F21
32.0
40.0 (min)
HPLC chromatograms of fluorous-tagged products showing (a) a clean separation and (b) a run in which three of the four compounds can be isolated in pure form. (Adapted with permission. Copyright 2001 American Association for the Advancement of Science.)
The whole proteome picture
1 2
3 4
5 6 7 8
Although the Saccharomyces cerevisiae yeast proteome has been widely studied, only 279 proteins have been identified to date. Now, John R. Yates III from Scripps Research Institute and Syngenta Agricultural Discovery Institute and his
9 10
Syngenta colleagues identify a whopping 1484 proteins. This catalog includes some proteins that are not usually observed by other methods: integral membrane proteins; proteins with molecular weights 190,000 Da;
CO2NH3+
Peptide mapping of the integral plasma membrane protein PMA1. Protein segments that were identified in this study (red) and transmembrane domains predicted by the Munich Information Center for Protein Sequences (MIPS) protein sequence database (cylinders) are shown. (Adapted with permission. Copyright 2001 Nature America.) M A Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
243 A
news
ANALYTICAL CURRENTS proteins with pIs 11; and low-abundance proteins such as transcription factors and kinases. To identify these proteins, the group optimized conditions for an established method that couples two-dimensional LC, in which a microcapillary column is packed with two chromatography phases, with MS/MS. The researchers analyzed
complex peptide mixtures from three fractions of an S. cerevisiae lysate. After each fraction was loaded onto its own microcolumn off-line, the columns were inserted into the instrumental setup, and no additional sample handling was required. Peptides were eluted in an iterative process in which the columns were reequilibrated and increasingly high con-
In vivo NMR A protein’s role can be predicted by its con-
in the cytoplasm of bacterial cells with in
formational state and location inside a cell.
vivo NMR. The researchers targeted MerA—a
Green fluorescent protein labeling is often used to determine cellular locations in vivo,
metal binding protein involved with the
but this technique gives no information on
detoxification of mercurials in E. coli. To
three-dimensional (3-D) structures. On the
separate MerA protein resonances from
other hand, NMR provides this 3-D informa-
other molecules within the cell, the pro-
tion, but to date, in vivo studies required the
tein was labeled with 15N by growing the
external addition of isotopes. Now, Volker
bacteria in 15N media. NMR spectra were collected using [15N,1H]-heteronuclear multiple quantum coherence
A
C
analysis. The authors show that the
100
E
NMR spectra are due to MerA in-
15N/ ppm
side the cell, rather than proteins outside the cytoplasm or bound to
120
the bacteria’s outer membrane. B
D
F
In vivo work will not replace in
100
vitro work, which provides structural information with easier preparation. However, studying a protein in
120 10.0
7.0
10.0
7.0
10.0
7.0
1H/ppm
its natural environment can give more information about protein in-
15
teractions, reversible small mole-
1
[ N, H]-heteronuclear multiple quantum coherence spectra of 15N-labeled MerA in various stages of isolation and purification. All experiments were measured on a 500-MHz NMR spectrometer at 37 °C.
cule binding, and posttranslation modifications. The researchers suggest that this concept can be extended to eukaryotic systems such as yeast, or to screen the
Dötsch and colleagues at the University of
membrane permeability of a potential
California–San Francisco describe a new
drug to a target protein. (J. Am. Chem.
and easier approach to examining a protein
Soc. 2001, 123, 2446–2447)
244 A
centrations of salt were added. The peptides were fed directly from the columns into an ion trap mass spectrometer equipped with a nano-LC electrospray ionization source. Like the chromatography and MS steps, the subsequent database comparison and identification of proteins were automated. For each class of protein, the number predicted agreed well with the number found. Thus, the authors conclude that their method is largely unbiased and is a step toward a comprehensive, automated, high-throughput system. (Nat. Biotechnol. 2001, 19, 242–247)
A N A LY T I C A L C H E M I S T R Y / M A Y 1 , 2 0 0 1
Raman studies SAMs Redox processes of self-assembled monolayers (SAMs) can be studied using microscopy and an electrochemical method such as cyclic voltammetry. However, this combination does not provide information about the molecular structure of the species involved. Time-resolved surface-enhanced resonance Raman (TR-SERR) spectroscopy does provide this crucial information; but to date, these studies have been carried out on bare silver electrodes, which tend to denature proteins. Peter Hildebrandt and Daniel Murgida of the Max Planck Institut für Strahlenchemie (Germany) instead conducted TR-SERR analyses of cytochrome c on a silver electrode coated with a SAM of 11-mercaptounadecanoic acid. Despite some attenuation of the surface enhancement, the researchers obtained SERR spectra that were very similar to spectra obtained for oxidized and reduced cytochrome c in solution. This is not the case for studies conducted on bare silver electrodes, in which the analysis is complicated by an additional conformational state. Thus, the researchers suggest that TR-SERR spectroscopy is a powerful approach to analyzing the dynamics and mechanisms of the interfacial processes of immobilized enzymes. (Angew. Chem., Int. Ed. 2001, 40, 728– 731)
news
Following dynamic changes Traditionally, spectroscopy has been the
capillaries, and the components are detect-
laser beam keeps the photochemistry lo-
tool of choice to follow fast dynamic sys-
ed individually at the outlet via 2-photon-
calized to the channel inlet. Moreover, the
tems; but Jason Shear and Mary Jane Gor-
excited fluorescence (Anal. Chem. 1999, 71,
multiphoton excitations occur periodically,
don of the University of Texas–Austin add
598 A–605 A). For example, using an 11-kV/
acting as an optical gate for the separa-
high-speed CE to separate and study multi-
cm field and a 2.8-mm capillary, a serotonin
tions. In one experiment, a 2-Hz fractiona-
ple components. They demonstrate the
photoproduct was electrophoretically trans-
tion yielded a spike from the optical gate
promise of this combined approach by rap-
ported through the capillary in ~50 ms.
idly analyzing the photochemical products of a serotonin metabolite and show that
every 0.5 s, followed ~170 ms later by a
The analytes continuously run through the CE capillary; however, a tightly focused
this method can characterize photoprodA key aspect of this technique is the
Capillary
Septum
use of multiphoton-excited photochemistry
tonin metabolite produced another photo-
Gate objective Sample solution
kV
ucts that degrade in milliseconds.
photoproduct peak. Adding a second sero-
Outlet buffer
tives from biological analytes” by hitting
Detection objective
the CE capillary. These packets are then quickly fractionated through very short CE
the spike. Because the data can be summed, this approach can be used even
Card
to create “packets of fluorescent derivathe analyte with laser light at the inlet of
product peak ~400 ms after
Schematic of the fast CE system. The CE capillary runs through a plexiglass card, and a rubber septum electrically isolates the two sides.
with molecules that have poor photoconversions or fluorescence properties. (J. Am. Chem. Soc. 2001,
123, 1790–1791)
“Shear” SECM know-how Researchers have begun to use scanning electrochemical microscopy with microelectrode tips to visualize the metabolism and redox activity of individual cells. However, the tips are kept at a fixed height, which does not accommodate variations in the tip-to-sample distances. Now, Wolfgang Schuhmann,
Andreas Hengstenberg, and colleagues at Ruhr-Universität Bochum (Germany) have a solution: Adjust the tip-to-sample distance using a shear force-based feedback loop. In this setup, the microelectrode vibrates, and a laser beam that is focused on the end creates Fresnel diffraction 45
70 65 60 z / µm 55 50 45
z / µm
40 35 30 25
80
60 60
40 y / µm
y / µm 40 20 0 0
20
40
60 x / µm
80
100
20
60 0
0
20
40 x / µm
Shear force-based topographic images of (a) a group of PC12 cells with a single cell in the front and (b) an individual PC12 cell. (Adapted with permission. Copyright 2001 Wiley-VCH Verlag GmbH.)
patterns that reveal amplitude and phase information. As the tip gets closer to the cell being analyzed, shear force dampens the vibrations and produces a phase shift. By continuously feeding these parameters into a software-controlled feedback loop, the tip can be kept at a constant distance of ~50–100 nm from the cell while the microelectrode detects changes in the concentration of redox-active compounds released by the cell’s metabolic and secretory processes. The researchers tested the method by monitoring the oxidation current during single vesicle exocytosis from PC12 cells. Although the method does not work with platinum microelectrodes that have been sealed in glass capillaries—the vibrating tip either shreds the cells or peels them off the glass substrate—it can be used with carbon-fiber microelectrodes that have been coated with insulating paint. (Angew. Chem., Int. Ed. 2001, 40, 905–908)
M A Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
245 A
news
ANALYTICAL CURRENTS Protein orientation sleuths
Mean height for: mAb1 + hST 2.44 2.25 nm mAb1 hST 1.85 nm
4 er, that method is sensitive to protein shape. To get around that problem, instead of 3 making their probes out of whole proteins, the Swedish 2 team used ligands that interact with particular protein re1 gions. These ligands were allowed to form complexes 0 0 500 1000 with the proteins on the sur3] Volume [nm face. Using AFM imaging, Volume versus height plot for a monoclonal antibody with the volume of the protein– added human serum transferrin. The mean height for ligand complexes was estieach population is marked. mated and compared with a prior estimate of the volume of the surface-adsorbed protein alone. The researchers note that protein transferrin as a probe, the rethe important parameter is the relative searchers studied the orientations of difincrease in volume, which indicates the ferent monoclonal antibodies against quantity of protein–ligand complexes transferrin. Further experiments, in formed, rather than the absolute volume which transferrin was adsorbed to two estimates. surfaces, revealed its orientation. (J. Phys. In two experiments using the human Chem. B 2001, 105, 2062–2069)
Height [nm]
What’s that protein up to now? Swedish researchers have proposed a Sherlock Holmes-like method to solve that mystery, using probes that track elusive protein placement. Magnus Bergkvist and colleagues at Mälardalen University and Uppsala University (both in Sweden) have come up with an atomic force microscopy (AFM)-based approach to study a protein molecule’s orientation by binding site-specific probe ligands onto surface-adsorbed proteins and measuring relative increases in protein volume. The researchers suggest that this method provides detailed information that can be helpful in manufacturing materials for bioimplantation or immunodiagnostic devices. AFM has already been used to analyze protein orientation in various ways, including the detection of a height increase when the protein on the surface is probed with another protein. Howev-
5
Detecting fuel traces in blood Methods that accurately assess human exposure to trace amounts of volatile organic compounds such as jet fuel are critical to public health. So Joachim Pleil of the U.S. Environmental Protection Agency and Siming Liu of ManTech
246 A
Toluene
Benzene
Environmental Technology, Inc., have been working in collaboration with the U.S. Air Force to modify a liquid–liquid extraction and GC/MS method to examine levels of JP-8—a fuel used in civilian and military planes—in whole human blood. Abundance Recent studies (a) 3000000 have indicated the ITSD toxic effects of 2000000 JP-8 on the skin, immune system, 1000000 postural balance, 0 pulmonary func30.00 40.00 50.00 60.00 70.00 10.00 20.00 Time Abundance tion, embryo 200000 (b) Decane Nonane growth, and kid3000000 150000 ITSD ney function. 100000 2000000 Undecane Breath tests are 50000 Dodecane the standard route Tridecane Tetradecane 1000000 Pentadecane 10.00 15. to determine ex0 posure, but they 30.00 40.00 50.00 60.00 70.00 10.00 20.00 Time are not as meanChromatograms of human blood (a) without JP-8 and (b) spiked with ingful as blood JP-8. (Adapted with permission. Copyright 2001 Elsevier Science.) content data. PreA N A LY T I C A L C H E M I S T R Y / M A Y 1 , 2 0 0 1
vious sample-preparation techniques for blood analysis did not yield high enough sensitivities to be valid, but a new preparation procedure developed by the researchers yields parts-per-trillion levels of volatile compounds. In the new method, blood is collected in a jar containing the anticoagulant heparin, diluted with a NaCl solution, extracted with pentane, and kept cold for transport. JP-8 levels are calculated from GC/MS data collected from dried and concentrated pentane solution with an internal standard. Researchers expect that this method will be used to monitor environmental and occupational exposure levels, and they hope to quantify and correlate these results to noninvasive breath tests, which can be used to study large populations. The method is believed to also be applicable to polyaromatic hydrocarbon and pesticide exposure. (J. Chromatogr. B 2001, 752, 159–171)
news
Tuning in to electronic molecular analysis Researchers in Texas are on a mission to fine-tune a new method for analyzing the electronic properties of molecules in selfassembled monolayers (SAMs). Allen Bard, James Tour, and co-workers at the University of Texas–Austin and Rice University show that it’s possible to rapidly characterize and screen molecules with differing electronic properties using a tuning fork-based scanning probe microscope and current–voltage measurements. The researchers say that the method shows promise for the high-throughput analysis of molecular electronic devices. In this method, the sharpened microDithering piezoelectric element Tuning fork STM tip (Pt) V SAM Au
I
A schematic of measurement and formation of the metal–molecule–metal junction with a tuning fork-based scanning probe microscope tip contacting the SAM on a gold surface.
scope tip is attached to a tuning fork, which is excited by a piezoelectric element oscillating at 33–100 kHz. A decrease in the amplitude and frequency of the oscillation indicates that the tip has made contact with the SAM, which has been deposited on a gold substrate. This permits a rapid approach to the surface. After contact is made, the potential across the SAM attains a characteristic bias voltage, and current begins to flow through the film. The magnitude of the current is a function of the conductance of the molecules. The tip can then be retracted slightly and moved to another location. The researchers tested the method on various SAMs and reproduced some previously observed phenomena, including a negative differential resistance effect on a 2´-ethyl-4,4´-bis(phenylethynyl)-1benzenethiolate SAM. In addition, during the analysis of a SAM containing dinitro substituents, the researchers saw distinctive current peaks in the negative bias region, which are usually attributed to the multielectron redox activity of dinitro compounds. (J. Am. Chem. Soc. 2001, 123, 2454–2455)
Organic fluorine in humans Organic fluorochemicals have been terrific for commercial applications, but their increased use since the 1980s raises questions about the levels of fluorine in humans. At 3M, Kristen Hansen and colleagues have developed a method of quantitative detection by negative ion electrospray MS/MS that quantifies specific fluorinated organic compounds in biological matrixes. The new method includes extraction with an ion-pairing reagent and analysis of the extract by HPLC and electrospray MS/MS. With this method, four organic fluorochemicals—perfluorooctanoate, perfluorooctanesulfonate, perfluorooctanesulfonylamide, and perfluorhexanesulfonate—were identified and quantitatively analyzed from the sera and liver tissue of 65 people. These studies yielded estimates of 27 ng/mL of total fluorine, which compares well with the earlier estimates of 26 ng/mL. (Environ.
Two species, or not two species? Anyone who studies lateral diffusion at chemical interfaces such as biological membranes and chromatography systems will be interested in knowing that single molecule spectroscopy can differentiate a system that has two different diffusing species from a system in which a single species is undergoing both diffusion and strong absorption. Mary Wirth and Derrick Swinton at the University of Delaware examined lateral transport of individual oligonucleotides (oligos)—made from the SP6 promoter sequence labeled with tetramethylrhodamine—at an interface of water and silica modified with chlorodimethyloctadecylsilane, which is the preferred stationary phase for HPLC
Sci. Technol. 2001, 35, 766–770)
analysis of oligonucleotides. Unlike fluorescence correlation spectroscopy, this method resolves radial trajectories of species passing through the beam. The count rate (I ) depends on the radial position (r) of the molecule in the beam. I varies with r for diffusing molecules, but I is constant for strongly adsorbed molecules. The researchers considered various models to explain the SP6 data, including beam distortion and self-hybridization of SP6. After removing long bursts, the researchers determined that the best fit for the remaining data was the fast lateral diffusion of a single species. The long burst data were best explained by strong, temporary adsorption of ~10%
of SP6 molecules to the surface—a phenomenon that produces the ubiquitous peak tailing of hydrogen-bonding species in HPLC. Assuming that the strong adsorption is due to hydrogen bonding of the oligo’s bases to the surface, not to adsorption of the fluorescent label, the results have implications for hybridization. The researchers note that oligos captured by the surface would undergo fast diffusion, which would facilitate hybridization, but the strong adsorption would lower the hybridization rate by ~10%. (J. Phys. Chem. B 2001, 105, 1472–1477)
M A Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
247 A
news
ANALYTICAL CURRENTS Total-analysis “taste” chip Are electronic cravings next? First we had electronic “noses”. Then came electronic “tongues”. Now, John McDevitt and a host of colleagues at the University of Texas–Austin bring us a chemical/optical array-based detection system that they call a total-analysis “taste chip” sensory system. The system is based around a silicon wafer that is dotted with inverted pyramid cavities, which serve as tiny reaction vessels and analysis chambers. Minuscule holes in the cavities allow reagents to
flow through. The array elements are microspheres made from polystyrene– poly(ethylene glycol) or agarose that sit in the cavities. The microspheres are labeled with various indicator molecules— for example, fluorescent tags, enzymes, or proteins—and are individually addressable. Once the silicon wafer is loaded with microspheres, the unit is sealed in a customized flow cell that allows optical detection. A charge-coupled device collects absorbance and fluorescence signals for rapid digital analysis. The system was tested by analyzing A pH, metal cations, sugars, and antibodies from beverages, biological samples, and other fluids. In addition, the researchers evaluated the system’s reproB ducibility, reversibility, concentration thresholds, and response times in static and flow-based experiments. The data acquisition rate was 30 Hz, and the site-to-site optical C1 C2 C3 C response variance was 2–4%. Now, the reR1 searchers say they are developing “theme” chips for R2 viral agents, bacteria, DNA, redox-active compounds, and other analytes. This R3 may lead to the development of “programmable” taste chips, in which speSchematic of antigen–antibody assay. (a) Antigens are bound to cialized beads are anbeads. If a solution containing the corresponding antibody is prealogous to software sented, antigen–antibody complexes form. (b) A fluorescently lamodules. (J. Am. beled secondary antibody is added. (c) Experimental results. Only Chem. Soc. 2001, 123, 2559–2570) the third column contains beads labeled with the proper antigen. 248 A
A N A LY T I C A L C H E M I S T R Y / M A Y 1 , 2 0 0 1
Cleaving digestionresistant proteins Coercing proteins to yield their secrets is a time-consuming process of purifying and identifying their digested fragments. The process is particularly cumbersome when proteins resist digestion. Steven Bark and colleagues at the Scripps Research Institute present a new tactic that overcomes many formerly nondigestible proteins in a short time and requires fewer processing steps. Noting that mobility is a key factor in proteolysis and that proteins are more flexible at higher temperatures, the researchers find that digesting proteins at 65 °C speeds up cleavage and conquers resistant structures. The secret is to use the protease thermolysin, which prefers to operate at high temperatures. No extra sample preparation or purification is required because the substrates are thermally denatured. After cleavage, the samples are analyzed by MALDI-TOF MS or LC/MS/MS, with only a single on-plate washing step to remove salts before MALDI. The researchers digested bovine serum albumin in
