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ANALYTICAL CURRENTS
Soft landings In any sequential combination of analytical techniques, MS invariably falls at the end of the line because the method uses so little sample, and what it uses it destroys. But what if it was possible to retrieve the sample after running the mass spectrum? Richard D. Smith and colleagues at Pacific Northwest National Laboratory describe a method in which samples are analyzed by FT ion cyclotron resonance (ICR) and then "soft landed" on a surface for further analysis. In the researchers' system, a 40-in.long probe with a soft landing surface at one end is coupled to a 7-T electrospray ionization (ESI) FTICR instrument. The probe is positioned to collect the ions ejected after mass analysis and then removed without disrupting the vacuum in the spectrometer. To demonstrate the approach, a 160mer double-stranded DNA sample was mass analyzed and then soft landed onto a nitrocellulose membrane. This cycle of soft landings was repeated 300 times, increasing the amount of sample. The landing membrane was then collected, and the trapped DNA was subjected to polymerase chain reaction (PCR) chemistry. A comparison of the amplified DNA with a control showed that the 160-mer DNA had been successfully collected. A second experiment demonstrated that a mixture of two 50-mer single-
Rapid breast cancer analysis The mutations that turn a normal cell into a malignant one are marked by changes in the expression of cellular proteins. Recognizing those changes could be the basis of a screening procedure for certain cancers. David M. Lubman and colleagues at the University of Michigan, Detroit Medical Center, and Wayne State University demonstrate that HPLC-generated protein profiles from whole-cell lysates of human breast cancer cells can be used to detect elevated levels of cancer-
strand oligonucleotides, which differed in mass by only 40 Da, could be separated and one of the DNA segments selectively soft landed. The stored waveform inverse Fourier transform (SWIFT) protocol was used to select the lower weight component ions for soft landing. On the basis of subsequent gel electrophoresis results the authors found that approximately one amol of material was retrieved from the membrane in inis case, i nis apnroach could aid in rt.
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(a) Negative ion ESI-FTICR of two 50-mer single-strand DNAs with charge states of 17- to 13-, (b) the mass spectrum of the SWIFT-selected DNA, and (c) the mass spectrum of the softlanded DNA (S) following PCR amplification, which now also includes the single-strand DNA's complement (C).
related proteins in malignant cells. In this study, two malignant cell lines were compared with a normal cell line. The cell lines originated from a single fibrocystic patient, thereby avoiding the normal expression variations found among unrelated individuals. The protein profile was collected on nonporous, Clg-coated reversed-phase LC columns. An aveeage of 50 ug of protein was injected for each HPLC run, which required 15-30 min. To identify the peaks, fractions were collected, and the separated proteins' molecular weights were determined by MALDI time-of-fllght (TOF) MS. Peptide
maps were also obtained from these fractions by taking each sample through a tryptic digest and pulsed delay extraction MALDI-TOFMS analysis; the peptide masses were then matched to proteins by searching a database. The authors report finding several key cancer-related proteins, such as p53, c-src, and h-ras, at highly elevated levels in the malignant cell chromatograms. Interestingly, there were also distinct differences in protein expression in the two malignant cell lines despite the common genetic background. (Rapid Commun. Mass Spectrom. 1999,13,1808-12)
Analytical Chemistry News & Features, December 1, 1999 787 A
