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ANALYTICAL CURRENTS Malaria protein by MS The recent publication of Plasmodium falciparum’s genome (the deadliest malaria parasite) brings to light the need for information on more than just genes. In particular, attempts to develop a vaccine for protecting humans from P. falciparum will most likely need to focus on more than one method of action because different proteins are produced at different stages of the parasite’s complicated life cycle. Hendrik Stunnenberg, Matthias Mann, and colleagues at the Nijmegen Center for Molecular Life Sciences, the Leiden University Medical Center (both in The Netherlands), the University of Southern Denmark, and the Wellcome Trust Sanger Institute (United Kingdom) have applied high-accuracy MS to analyze the proteins present in various stages of the malaria parasite’s life cycle. The parasites were cultured, lysated, and separated by centrifuge. The soluble portion that contained most of the parasite proteins and human red blood cell proteins was then separated into 10
mass fractions using one-dimensional gel electrophoresis. The bands were tryptically digested and separated by reversed-phase LC with quadrupole timeof-flight MS/MS (nanoLC/MS/MS). The process was repeated with the insoluble fraction, which contained many of the life-cycle-specific surface proteins. The resulting data, after iterative calibration algorithms, had a mass deviation of 0.03 Da for a tryptic peptide of 1300 Da. This fragment data was then compared with calculated fragment data for all tryptic peptides from human and parasite sequences. The result of the comparison and further analysis of the MS/MS data detected 1289 unique proteins for the parasite. The researchers report that 315 of the proteins were found exclusively in sexually reproducing parasites, and 226 were found exclusively in the asexual form. To highlight the findings, only three of the extensively studied Pfs48/ 45 sexual-stage surface antigens were found in the asexual-stage parasites,
Figure not available for use on the Web.
whereas 57 were found in the sexual form. (Nature, 2002, 419, 537–542)
Shotgun proteomics and low-level proteins
moFinnigan analyzed the plasma proteome. In particular, they targeted human growth hormone (hGH), which is expressed at low
Relative abundance
trap MS, Shiaw-Lin Wu, William Hancock, and colleagues at Ther-
239.92
100
To assess the dynamic range of “shotgun” proteomics using ion
(a)
80 60
233.25
40 20
276.19
175.05
0
0
50
100
150
200
In the shotgun approach, a complete protein sample is digested, and the peptides are resolved using chromatography and MS/
range of 1 in 40,000. (The samples were spiked so that the hGH levels were 10-fold above normal.) The researchers also compared various sample prefractionation protocols, ranging from no fractionation to two-dimensional chromatography. (J. Proteome Res. 2002, 1, 459–465)
100 Relative abundance
hGH was detected at the low-femtomole level with a dynamic
323.64
356.34 359.76
327.29
levels in a background of highly expressed proteins.
MS. More than 200 plasma proteins were identified in 8 h, and
249.99 273.36
194.80 199.43
hGH 142.95 128.26
250 Time (min)
300
504.20
440.51
350
400
450
500
548.15
550
472.3
(b)
80 60
686.4 392.3
40 245.1
20
236.9
0
150
200
294.1
250
300
346.0
350
431.0
400
456.7 473.3
585.3 556.4
450
500
550
600
668.3
650
696.4
700
750
800
850
900
m/z
(a) Elution profile of the peptides in a plasma sample spiked with human growth hormone (hGH). (b) MS/MS fragmentation of the first tryptic peptide (T1) from hGH. D E C E M B E R 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y
603 A
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ANALYTICAL CURRENTS
One way to simplify the screening of drug candidates is to use small amounts of material and only look at the ligands that bind tightest to their biomolecular target. Maria Wigger and colleagues from the University of Florida and the National High Magnetic Field Laboratory demonstrate this approach by using FT ion cyclotron resonance (ICR) MS and IR multiphoton laser dissociation (IRMPD) to selectively remove noncovalent ligands from biomolecular complexes in the gas phase and identify them. They find that this technique works well with high-affinity polar ligands but discriminates against hydrophobic molecules. The researchers tested their MS approach by screening a 324-member combinatorial peptide library against the hematopoietic cell kinase (Hck) SH2 domain, which is involved in important cellular signal transduction pathways. After exposing the domain to the library, they used electrospray ionization to gently generate gas-phase ions of the noncovalent complexes. To simplify the analysis, only the complex ions with a m/z of interest were stored in an ion trap. Then, IRMPD, adjusted to a laser power that dissociated ligands but did not fragment peptides or proteins, stripped the ligands from the Hck SH2 domain ions. Once free, the ligands could be identified by their mass spectrum. Only 1–2 nmol of protein was needed for an experiment. The researchers found two major peaks in this example, one of which was linked to a ligand known to have the highest affinity binding sequence for the closely related Src SH2 domain. However, screening the same 324-compound library against Hck SH2 using a conventional screening protocol discovered additional high-affinity ligands. The researchers report that these added ligands have a hydrophobic–hydrophobic binding motif. (J. Am. Soc. Mass Spectrom. 2002, 13, 1162–1169)
Figure not available for use on the Web.
604 A
A N A LY T I C A L C H E M I S T R Y / D E C E M B E R 1 , 2 0 0 2
“Smart” nanotubes above average in bioanalysis 200
N N
RS
SS
150 Abundance
No water necessary
N * OH *
N F
100
I II
50
III 7
8
9 10 Elution time (min)
11
Chiral HPLC chromatograms for racemic mixtures of the drug FTB (I) before and (II, III) after extraction with 18 mg/mL of 200-nm antibody-containing nanotubes.
Charles Martin and co-workers at the University of Florida–Gainesville and VTT Biotechnology (Finland) apply different functional groups to the inner and outer surfaces of silica nanotubes and demonstrate that they can make “smart” nanophase extractors to remove molecules from solution. The researchers used silica nanotubes that had been synthesized within alumina template membranes with 60- or 200-nm-diam pores and reacted the inner nanotube surfaces with the first silane while the nanotubes were still embedded within the template membrane pores. Because the outer surfaces were in contact with the pore wall, they were masked, and the silane did not attach to them. Then the researchers dissolved the template, freeing the nanotubes and exposing the outer surfaces. Next, they reacted the nanotubes with a second silane, which attached only to the outer surfaces. According to the researchers, nanotubes with hydrophilic chemistry on their outer surfaces and hydrophobic chemistry on their inner surfaces are ideal for extracting lipophilic molecules from solution. They demonstrated this by using such nanotubes to remove 82% of the compound 7,8-benzoquinoline from a 1 10–5 M solution. To illustrate a more general extraction capability, the researchers used antibody-functionalized nanotubes to selectively bind the RS enantiomer of a test drug. To prove that biocatalysis is possible, the researchers immobilized the enzyme glucose oxidase on silica nanotubes and obtained an activity of 0.5 ± 0.2 units/mg of nanotubes in a dianisidine-based assay. (J. Am. Chem. Soc. 2002, 124, 11,864–11,865)
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APCI-MS method improves explosives detection B. J. Keely and colleagues at the University of York and Mass Spec Analytical, Ltd. (both in the United Kingdom) introduce a rapid and efficient mass spectrometric method for detecting trace amounts of explosives under negativeion, atmospheric-pressure chemical ionization (APCI) conditions. They report that complexing samples with chlorine improved the limit of detection—in some cases by several orders of magnitude—and that a Hotblocks sample introduction system coupled to an ion trap mass spectrometer helps to lower costs. The researchers tested for trinitrotoluene (TNT), nitroglycerine (NG), pentaerythritol tetranitrate (PETN), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Keely and his team introduced samples into an APCI source via the heated-plate inlet of the Hotblocks system with a supplementary gas feed of dichloromethane. This improved the formation of the analytes’ chloride adducts and minimized the adducts’ decomposition during analysis. The researchers add that introducing the analytes via the Hotblocks interface, rather than a conventional APCI interface, forms fewer cluster ions. The experiments also expanded on previous work in which the researchers systematically optimized trapping parameters for an ion trap spectrometer to improve the reproducibility of the spectra and the detection of low-mass fragment ions. In particular, simplex optimization of MS/MS resulted in transitions to m/z 62 or 46, which were intense and suitable for recognizing NG, PETN, and RDX. Compared with electrospray ionization, APCI had better limits of detection for TNT, NG, PETN, and RDX, with an improvement of 3 orders of magnitude for TNT. (Rapid Commun. Mass Spectrom. 2002, 16, 1883–1891)
Figure not available for use on the Web.
The spliceosome analyzed In eukaryotes, the spliceosome has the job
matography to isolate spliceosomes and
of removing the “introns”, or noncoding re-
nanoscale microcapillary LC/MS/MS to
are believed to be involved in other gene-
gions, before a segment of RNA is ready
characterize the components, the re-
expression steps, such as transcription elon-
for translation into protein. This critical
searchers identified ~145 proteins. This
gation, polyadenylation, and mRNA export. In
“machine” is made from small nuclear
catalog makes the spliceosome the most
particular, every protein associated with the
RNAs and proteins but has never been fully
complex cellular machine characterized so
TREX complex, which is involved in transcrip-
characterized. Now, Zhaolan Zhou and col-
far. The list includes all of the previously
tion elongation, was found in the spliceosome
leagues at Harvard University and Harvard
known splicing factors and 58 components
in this study. This apparent dual functionality
Medical School present a comprehensive
that have not been associated with splicing
supports the idea that the cell’s various gene-
proteomic analysis of the spliceosome.
previously. The functions of 22 of those
expression machines are coupled to one
proteins are completely unknown.
another. (Nature 2002, 419, 182–185)
Using a combination of affinity chro-
At least 30 of the spliceosome proteins
D E C E M B E R 1 , 2 0 0 2 / A N A LY T I C A L C H E M I S T R Y
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ANALYTICAL CURRENTS DNA separations in seconds Separating DNA using traditional pulsed-field gel electrophoresis takes far too long, anywhere from 10 to 200 h. New methods, such as those based on flow cytometry or capillary gel electrophoresis, have reduced this to minutes, but now it may be down to seconds. Edward Cox and colleagues at Princeton University have developed a separation method that they compare to a prism, which uses microscale posts and channels on a microchip. The DNA solution is injected onto the chip and, in the presence of a uniform electric field, migrates through a hexagonally packed array of posts. The DNA is separated at different angles on the basis of length, akin to how light is separated by a prism. Although previous microfluidic chips have been developed to separate DNA, the researchers believe they have improved on the design by using the microchannels that deliver the sample and remove the DNA to also act as electrodes. This reduces the production of bubbles and provides a more uniform electric field. Also, the device can separate DNA in a continuous-flow operation, something previous microfluidic chips did not allow. The concept was tested using bacterial artificial chromosomes and P1-derived artificial chromosome inserts. Four chromosome inserts, ranging from 61 to 209 kb, were separated using field strengths of 20–250 V/cm and pulse durations of 10–500 ns.
Figure not available for use on the Web.
The varying conditions produced different results because small molecules migrated along the average field direction, whereas larger molecules migrated in the strong pulse direction, so pulse and field changes tuned the separation. The prism separated the DNA in approximately 15 s, or about 1000 times faster than traditional gel electrophoresis. In addition, the researchers report that the method has ~13% resolution, which puts it ahead of other microfluidic DNA separation techniques. (Nat. Biotechnol. 2002, 20, 1048–1051)
Unstable pigments in the U.S. dollar The precision and detail of the technology being used to make counterfeit money today, right down to the pigments in the dyes, makes distinguishing between authentic and forged monies especially daunting. But V. Rusanov, K. Chakarova, and T. Madolev of Sofia University and the American College of Sofia (both in Bulgaria) are using Mössbauer spectroscopy to more accurately differentiate between authentic and forged U.S. currency on the basis of the stability of the pigments. Previous studies concluded that specific iron-containing pigments are often used during the printing of certain currencies. In the current study, the researchers used the sensitive Mössbauer spectroscopy technique to look at the stability of iron-containing pigments in 54 authentic $100 bills printed between 1934 and 1993 and compared their values with those of 10 “high-quality” counterfeit bills. The black pigment (a mixture of Fe3O4 and -Fe2O3) was shown to be very stable and consistent, whereas the green pigment, which contains Fe3+, was shown to be highly inconsistent in all 10 forged bank notes. Normally, such instabilities in the dye pigments allow authorities to distinguish between authentic and forged bills. However, the Mössbauer spectra showed inconsistencies among the green pigment spectra for the authentic notes and revealed the total lack of Fe3+ pigment in 8 of the 54 genuine bank notes. Because of this inconsistency, Rusanov concludes that to accurately determine counterfeit from authentic monies, it’s important to look at multiple parameters, rather than just a few, using a sensitive method such as Mössbauer spectroscopy. (Appl. Spectrosc. 2002, 56, 1228–1236) 606 A
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Figure not available for use on on the Web.
