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ANALYTICAL CURRENTS Molecules walk the nanowire (a) S
NanoFET NW D
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Nanosensor NW D
Backgate NH3+ Si H H OOOOO
NH2 Si H H OOOOO
NH2 Si
O–O O O O– + + – H – H Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si SiNW SiNW SiNW + + +H +H
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Conductance (nS)
Nanowire field effect transistors (FETs) are very sensitive detectors, mainly because their “bulk” is so small that the influence of even a single molecule is felt. For Charles Lieber and his associates at Harvard University, the ideal material for these FETs is silicon because it allows sensitive tuning and convenient chemical modification of the oxide surface to create a wide range of chemical and biological sensors. The first in a “palette” of label-free nanosensors created by the researchers is a 3-aminopropyltriethoxysilane-modified silicon nanowire that detects pH with good, reproducible results over a pH range of 2–9. Next is a biotin-functionalized nanowire that can detect streptavidin down to the 10-pM level, which means that it is more sensitive than stochastic sensing of single molecules, the researchers say. A similar real-time antibody detection device is based on the
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(a) Poly(dimethylsiloxane)–silicon nanowire pH sensor. The source (S) and drain (D) are shown. (b) Linear relation between pH and conductance. (Adapted with permission. Copyright 2001 American Association for the Advancement of Science.)
binding of antibiotin to biotin. In this case, the binding is reversible, unlike the biotin–streptavidin binding, and the sensor’s response reflects the concentration of antibiotin. Finally, there is a calcium nanosensor
in which Ca2+ binds reversibly to immobilized calmodulin. The researchers suggest that these and other nanosensors might be useful for array-based screening and in vivo diagnostics. (Science 2001, 293, 1289–1292)
Little bits make color Nicholas Kotov and colleagues at Oklahoma State University and the Hahn-Meitner-Institut (Germany) may have found a pot of gold with the nanosized rainbows they have created. The researchers demonstrate
how to use layer-by-layer assembly (LBL) of nano-sized particles, often referred to as quantum dots, to produce one-dimensionally graded semiconducting films. Although gradation and ordering of media has improved the performance of optical and electronic devices, graded semiconductors remain difficult, expensive, and hazardous to make by conventional chemical vapor deposition or molecular beam epitaxy methods. LBL is versatile and simple: Water-soluble nanoparticles are layered on a substrate in order of increasTransmission electron microscopy images of ing size. In this case, the assembly of cross sections of a graded film made from colored luminescent CdTe nanoparticles five bilayers of green, yellow, and red nanoparticles at different magnifications. (with a polyelectrolyte) range from green
for the smallest particles through yellow and orange to red for the largest, with 5–10 bilayers for each color. This ordering of layers is key to optimizing the materials. Transmission electron microscopy was used to examine cross sections and evaluate the internal structure of the film. Confocal optical microscopy also established the graded nature of the film. Although the graded approach has some physical limitations related to the film roughness, combining particle-size quantification and gradation provides various new optical and electrical effects from materials based on charge transfer. (J. Am. Chem. Soc. 2001, 123, 7738–7739)
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ANALYTICAL CURRENTS Deuteriums in unexpected places Exchanging deuteriums for hydrogens (m-isophthalic acid; see figure) or the where they underwent H/D exchange in proteins and other biomolecules is a methyl hydrogens (2-methylisophthalic) with either D2O or ND3. With a small amount of excess internal energy (10 s), which is much longer than the uptake time (~100 ms) for the fluorescent dyes. (Anal. Biochem. 2001, 295, 138–142)
new vapoluminescent platinum(II) salts. They test their device on several solvents of environmental interest. Salts are considered vapoluminescent or vapochromic if they emit particular wavelengths in response to contact with a specific type of solvent vapor. In the new array, each salt contributes bits of information that are taken together to identify the detected solvent. The mechanisms are not completely understood, but the first salt’s luminescence intensity increases, whereas the second salt’s intensity decreases as luminescence maximums shift to shorter wavelengths. The third salt’s emission is blue-shifted for hydrocarbons and red-shifted for chlorinated hydrocarbons, alcohols, and water. The researchers deposited submilligram quantities of the salts onto inert platinum or carbon fiber disks. Following exposure to solvent vapors, principal component analysis helped to identify the analytes. (J. Am. Chem.
The Institute of Analytical Sciences, LYON (France) Director Position Within a project co-sponsored by the State and the Rh ne-Alpes region, the CNRS and the University Claude Bernard, Lyon-I, are currently -seeking a Di rector whose aim will be to create, launch and manage an internationally rec ognized Institute of Analytical Sciences. This Institute will consist of a number of research teams, some - of them re sulting from existing laboratories (150 people), some to be created but also others resulting from strong collaboration with various partners such as CEMAGREF or CPE Lyon. The goal of the Institute is to be an international centre for: ¥ Research in analytical methods ¥ Analytical services ¥ Expertise with a National and European reputation ¥ Training in new technologies and methods in analytical sciences at the graduate and undergraduate level ¥ Research valorisation through entrepreneurship Its research, ranging from analytical chemistry to analytical biochemistry, will be in the following areas: ¥ Food science ¥ Materials analysis ¥ Pharmacology, analytical toxicology and proteomics ¥ Environment ¥ Instrumentation, analytical methodologies and computer science Applicants should have a world-class reputation in the area of- analytical chem istry and a strong knowledge of industrial problems. People from industry are welcome to apply. Strong management and leadership skills are required since the Institute will have to launch and conduct major international projects in re lation with industry as well as local, national and European government agen cies and universities (research and teaching organisation). This will require the construction of a scientific, technical, administrative and financial structure. The ultimate goal for the Director is to make this Institute the centre of excellence for European research in analytical sciences. Further information can be obtainedProf. from J. Remillieux (Universit Claude Bernard, Lyon I), tel: 33 (0)4 72 43 12 82, e-mail:
[email protected]. Application deadline date is December 10, 2001. Please send resume and covering letters to:
Soc. 2001, 123, 8414–8415) N O V E M B E R 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
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RESEARCH PROFILES An unfolding protein tale
Ion signal intensity
intensity of each multiply charged mopeaks. Although the intensities of these Finding a facile method to monitor the lecular ion peak is proportional to the peaks changed with pH, the central valhabits of proteins in solution has been number of protein conformers in soluues and half-widths of these peaks did the thrust of much recent research. tion with surface areas capable of stabinot. A spectrum could be represented Could it be that all we had to do was lizing that charge. In this way, an obby a linear combination of charge-state step back and get a fresh perspective on served ESI mass spectrum can be data that has been right in front of us all distributions, called “basis functions”, considered a sum of the contributions which could be approximated as Gaussalong? It seems to be so. from each protein conformer. ian distributions. The intensity changes In the October 15 issue of Analyt“We can pick up contributions from could be represented by a weighting ical Chemistry (pp 4763–4773), Igor every single conformer [of apomyogloKaltashov and Andras Dobo at the Uni- factor, which accounted for the relative bin],” Kaltashov says, “and follow its contributions to the overall charge-state versity of Massachusetts–Amherst deevolution as a function of pH.” distribution. scribe a new way to look at protein MS. However, the ␣-helix is just one type For the myoglobin data, the reIn the process, they convert an ordinary searchers used a standard software pack- of secondary structure. To demonstrate mass spectrometer, run in a standard the applicability of this ionization mode, and offtechnique to other classes the-shelf software into a I exp(n) of proteins, Kaltashov apsemiquantitative monitor Icalc(n) I(n) = ΣbiBi(n) plied the same experimental of protein surface areas, strategies to cellular retinoic and they get an extraordiBasic function Bi (n): acid binding protein— nary view of protein con– (n – ν0i)2 2σ1 a representative of the former dynamics in multi1 2 2σ Bi(n) = e -sheet protein class. component solutions. 2 2πσ Again, previous work had To test the new ap2 σ2 suggested that four conproach, the researchers 0 - population central; νi 2 formers exist. Kaltashov chose the apo- and holo0 0 σ - population variance ν1 ν2 found that four basis funcforms of myoglobin, beIon charge state, n tions gave the best fit to the cause myoglobin is a wellIexp(n) Linear optimization: Icalc(n) data, and conclusions about behaved representative of the spread and central valESI MS data is converted to charge state versus intensity to calculate the ␣-helical proteins and the ues of these basis functions basis functions corresponding to protein conformers in solution. subject of a large body of corresponded well with prior research. The popular the published findings. belief is that, at neutral pH, A change in temperature or addition age to fit curves and calculate the cenapomyoglobin presents only one stable of a ligand can also evoke non-native tral values and half-widths of the basis conformer in solution. “[But] we are protein conformations. “We did acid showing that there are four different con- functions. The researchers initially ran unfolding in this work, but obviously the calculations using three basis funcformers of apomyoglobin in solution at this technique has much broader appliany given pH we studied,” says Kaltashov. tions, but the best results came when cations,” says Kaltashov. This finding results from a new analy- the researchers used four basis funcKaltashov’s approach may seem a tions. On the basis of these results, as sis of electrospray ionization (ESI) MS road less traveled to the classically data. ESI MS of proteins typically gener- well as the information available about trained chemist or biochemist, and that myoglobin, the researchers concluded ates a wide range of multiply charged is for good reason. “To tell you the that they were seeing all four known molecular ion peaks; clever researchers truth, I was trained as a physicist initialconformers of apo- and holomyoglobin often use this phenomenon to extend ly, and with that training came an extenduring unfolding. the mass range of standard MS instrusive background in mathematics,” he According to Kaltashov, “By unfoldmentation, giving more emphasis to the ing the protein, we increase the solvent- says. His unique background provides a m/z coordinates than the intensity axis. accessible area.” The more a protein is fresh new view of a familiar observation. Kaltashov realized that the ion peak inunfolded in a buffered solution, the And when a different perspective results tensities in this mass spectral region dein such an elegant solution, that is great scribed the digitized envelope of a curve, more surface area is available to be protonated and the higher the charge its reason to hail diversity. a which appeared to be formed by a com—Zelda Ziegler ion will have during ESI MS. The signal bination of a few smooth, overlapping
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Elusive membrane proteins: The next chapter It’s trite but still true that the part doesn’t equal the whole, especially for mass spectrometrists who are trying to find out what proteins really do in a cell. Developments in MS over the past few years now allow researchers to take small snippets of proteins and churn out identities at rates they once only dreamed of. But to discover crucial behavior—how proteins send messages and what they control, for example, “you have to be able to observe the entire protein,” says Daniel Knapp of the Medical University of South Carolina. In the October 15 issue of Analytical Chemistry (pp 4774–4779), he and his colleagues report what they believe is the first complete MS mapping of a recombinant integral membrane protein. Their ultimate goal is to find how a protein’s chemical and physical structure directs functions within a cell. Although Knapp describes the work as simply an incremental improvement in methodology—“the result of working at it and getting the right combination of steps and the appropriate care and handling”—the process is able to analyze native or expressed proteins that are present at low concentrations. “We’ve been able to adapt our methods for analysis of these integral membrane proteins to smaller amounts,” he says. “And while this smaller amount that we report in this paper is not small at all compared to what people are doing with other soluble proteins, it’s significant in the case of integral membrane proteins because they’re much more difficult to deal with analytically.” The researchers believe their method for studying subnanomolar samples is useful for determining proteins that couldn’t be previously analyzed. In previous work with native rod cell membranes from the eye, the South Carolina group had reduced and alkylated cysteine residues still in the membrane. Although a significant amount of the sample was lost in the experimental workup, there was still sufficient rhodopsin
for electrospray ionization (ESI) MS and complete sequencing of the protein. This was possible because the predominance of rhodopsin in the native rod cells means that the protein is very purified to start with, explains Knapp. But this new method deals with recombinant bovine rhodopsin expressed from cone outer segment cells, he says, and the protein represents a very small proportion of the total membrane protein. Therefore, the recombinant protein has to be removed from the membrane and purified on an antibody affinity column. But the researchers found that, because of denaturation, the MS signal decreased if they reduced and alkylated proteins after removal from the membrane. A detergent saved the day, removing proteins from the membrane without complete denaturation. “We found that we could actually carry out some of that chemistry that we were doing in the membrane . . . in detergent solution,” says Knapp. Cyanogen bromide cleavage and HPLC prepare samples for MS analysis. ESI with tandem MS identified the pri-
mary protein structure and all but four types of posttranslational modifications. The disulfide bridge and a retinal chromophore attached to lysine were cleaved and completely lost. A doubly glycosylated fragment and an acetylated methionine fragment also were lost to ESI MS. However, the researchers did observe the double glycosylation and a small number of acetylation sites using MALDI MS on fractions split from the HPLC column. “There are few approaches that do everything,” he notes. Knapp says that there hasn’t been much in terms of good methodology for MS analysis of integral membrane proteins, so their functional studies have been somewhat “stymied”. Now, the researchers are looking forward to using their new tool for probing protein structures by cross-linking and other chemical modifications. “For example, one of the things we’re trying to do is to find out how the shape of the protein changes in these signal receptor proteins . . . [during] the signal reception event,” he says. a —Judith Handley
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Amino acid sequence of bovine rhodopsin. Numbers label the first amino acid of the cyanogen bromide cleavage fragments, and broken circles indicate fragments detected by MALDI MS. N O V E M B E R 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
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RESEARCH PROFILES Researchers brainstorm new microdialysis method Robert Kennedy of the University of Florida had faith. He had heard other researchers talk about obtaining fascinating mass spectra with low-attomole sample quantities, and he believed such spectra might be used to identify living brain chemistry—in this case, neuropep-
Capillary LC columns with integrated electrospray emitters. (a) Bright-field optical image of the column end with emitter tip. (b) Scanning electron microscopy (SEM) of macroporous frit formed by in situ photopolymerization. (c) SEM of electrospray emitter with end-on view. 590 A
tides collected in vivo. In February, his hopes were answered. Graduate student William Haskins showed excited onlookers the mass spectra of peptides with high S/Ns at picomolar concentrations using a new microdialysis method. “Everybody in our group was really amazed because we’d been trying to detect picomolar concentrations of peptides with many different methods, and we were getting little signals here and there,” Kennedy recalls. “[Haskins] just walks in, and he’s got this great signal– noise ratio of these things.” In the November 1 issue of Analytical Chemistry (pp 5005–5014), Kennedy and colleagues describe how they use miniaturized columns with electrospray emitters, optimized gradients, and quadrupole ion trap (QIT) MS to monitor and discover endogenous neuropeptides in vivo from the globus pallidus region of the brain in male Sprague–Dawley rats. The technique was coupled on-line to microdialysis sampling, and the sensitivity allowed monitoring of endogenous neuropeptides at basal and stimulated levels with at least 30-min temporal resolution. “We’re getting a full mass spectrum, which gives you better identification than single ion monitoring,” says Kennedy. “We can monitor these peptides, we think, reliably on a good timescale.” Neuropeptides are an important group of interneuron-signaling molecules that act as hormones, neuromodulators, and neurotransmitters. Scientists don’t know all the transmitters being made in the brain, and they’re not sure what neurotransmitters are involved in many different behaviors, says Kennedy. For example, what happens in the brain when one becomes sleepy? “You’ll never know about the [underlying chemical] behavior from looking at single neurons or tissue sections, which is why in vivo methods are necessary,” says Kennedy. “This new method may eventually allow us to not only monitor known peptides for their role in behavior but also [to]
A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 1
identify [the] novel neurotransmitters or unexpected peptides involved.” Neurons that release neuropeptides start off by making proteins—such as proenkephalins present in the brain, spinal chord, and gut—and use enzymes to break them down. The problem is that these neuropeptides are hard to measure because of their low concentrations in microdialysis samples, explains Kennedy. “We knew that smaller columns and electrospray tips would provide better sensitivity, but we didn’t know how small we could make it be reliable,” he adds. Kennedy’s team interfaced 25-µm i.d. fused-silica capillary LC columns with 3-µm-i.d. integrated electrospray emitters to a QIT mass spectrometer. The researchers constructed the column by making frits through in situ photopolymerization of glycidyl methacrylate and trimethylopropane trimethacrylate. They then pulled the column outlet to a fine tip with a CO2 laser puller to prepare the electrospray emitter. Finally, they slurry-packed the column with 5-µm reversed-phase particles. They used large-volume injections with an automated two-pump system that allowed high flow rates for sample loading and low flow rates for elution. The group achieved a detection limit of 4 amol, corresponding to 2 pM for 1.8 µL injected on-column for a mixture of peptides dissolved in artificial cerebral spinal fluid. Time-segmented MS2 scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. One unknown peptide identified was tagged Peptide I1-10 (SPQLEDEAKE), a product of preproenkephalin. Kennedy is confident there are more compounds to be found at lower levels, and he hopes to achieve even higherquality spectra. “I think that understanding the brain is probably one of the biggest challenges facing scientists,” he says. a —Cheryl M. Harris
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MS for carbohydrates
Relative abundance
the picomolar range and (a) BS 100 allows quantitation by en48% suring that peak height is indicative of abundance yet independent of the B size or the monosaccha50 26% ride composition of the BS2 26% oligosaccharide. The authors first applied the method to re0 combinant soluble CD4, 20 40 0 10 30 min which has two N-glycosy(b) lation sites. They analyzed 1314.8 BFS2+ BS2+ 1227.8 100 the released glycan pool simultaneously by stanBF22+ 1134.1 dard HPLC of reductively B2+ 1047.1 1408.5 BS22+ aminated oligosaccharides BFS22+ and by electrospray MS of 50 1495.5 permethylated derivatives. Both approaches showed B+ BF+ BS+ + that the glycans had two 2071.3 2245.3 2432.7 BFS 2606.7 BS2+ antennae and varying de2794.0 0 grees of sialylation and 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 m/z fucosylation. However, HPLC was able to resolve Comparison of HPLC with electrospray MS of glycans the mixture into only released from soluble CD4. three clusters, whereas MS showed that six species information about the relative proporwere present (see figure). This was betions of the various linkages. cause MS was able to distinguish the Domon notes that the method won’t fucosylated and nonfucosylated strucpoint out the precise location of the tures, says Domon. On the other hand, glycans on the protein, but this can be HPLC could only distinguish species determined by first cleaving the protein based on their sialic acid content. into peptides and analyzing the glycoNext, the researchers used the same sylation pattern of each peptide. Also, parallel approach on a protein with a more complex glycosylation pattern, the the method cannot distinguish between structures with the same mass, such as ␣1-acid glycoprotein. Here again, MS glycans that differ only by the location resolved more components among the of the sialic acid(s) within the molecule. heterogeneous mixture of bi-, tri-, and And, although this method provides tetraantennary glycans carrying various better structural information than connumbers of sialic acids, fucosyl residues, ventional HPLC, it requires more time. and lactosaminyl units. By desialylating This is partly because, with the tedious the protein before releasing the N-glydata processing, sample preparation cans and then applying their method, takes longer. “Right now, there’s no the researchers could quantitate the good algorithm to get the deconvolulevels of components present in even minute quantities, according to Domon. tion automated,” he says. “But ultiBy using neuraminidases to specifically mately, it’s worth the effort since you cleave different types of sialic acid linkgain much more information.” a —Alka Agrawal ages, the researchers also could obtain Relative abundance
More and more often, recombinant glycosylated proteins are used as therapeutic agents. However, glycosylation can vary, which may alter an agent’s biological activity. To characterize such sample heterogeneity and ensure batch-to-batch consistency at all stages of production, researchers need robust, rugged methods to determine the glycosylation patterns of proteins. A common way to analyze protein glycosylation is by releasing the glycans from the protein, labeling them with a fluorophore such as 2-aminobenzamide, and analyzing these derivatives by HPLC. This approach, based on the retention time of the detected species, provides quantitation but only limited structural information about the glycans. In the October 15 issue of Analytical Chemistry (pp 4755– 4762), Bruno Domon (now at Celera Genomics) and his colleagues at Biogen describe a new semiquantitative method that analyzes the glycosylation pattern of proteins and provides structural information. The method involves the enzymatic release of the N-linked glycans, the permethylation of the glycan pool, and the analysis of the products by either electrospray or MALDI MS. The method is highly reproducible, with variability of
