Offering direct detection of biomolecules, matrix-assisted laser desorption/ionization (Maldi) recommends itself for a wide variety of applications, including diagnostic applications involving detection of specific biomarkers through the direct analysis of diseased tissue. Some classes of biomolecules — oligonucleotides or sugars, for example — do not lend themselves to direct analysis by Maldi, however. This difficulty is the result of the large size and low abundance of the molecules in cells as well as of analytical difficulties deriving from the presence of certain phosphate groups.Researchers have reported a technique that allows indirect detection of oligonucleotides and other classes of biomolecules that do not otherwise lend themselves to direct detection by Maldi. The method couples a small, easily detected molecule of known mass to the Maldi probe with a photocleavable linker. Laser irradiation during Maldi releases the molecule, thus enabling imaging. Courtesy of Isabelle Fournier, Université des Sciences et Technologies de Lille, France.Researchers from Université des Sciences et Technologies de Lille in Villeneuve d’Ascq, France, from Société Eurogentec in Seraing, Belgium, from the National Institutes of Health in Baltimore and from Université de Sherbrooke in Quebec, Canada, therefore set out to develop indirect detection of oligonucleotides using Maldi. In a paper published in the June issue of Journal of Proteome Research, they described their technique and how it can be extended to detect any kind of molecule.Dubbed “Tag-Mass,” the strategy takes advantage of a small molecule of known mass that is easily detected using Maldi mass spectrometry. The molecule is coupled to a probe molecule using a photocleavable linker that has an absorption wavelength (340 nm) close to that of Maldi lasers (337 to 355 nm).Laser irradiation during Maldi induces cleavage, thus releasing the tag molecule, which can then be imaged. The method facilitates indirect detection of biomolecules that are otherwise inaccessible or barely accessible by Maldi. A number of classes of probes are compatible with the tagged photocleavable linkers, including DNA, cDNA, single-stranded cRNA and antibody.Mass spectrometryThe researchers assessed the efficacy of the method on slices of rat brain, using several oligonucleotide and antibody probes. They performed Maldi-Tof mass spectrometry using a mass spectrometer made by Applied Biosystems of Framingham, Mass., with delayed extraction and a 337-nm pulsed nitrogen laser, and imaging using a Sony color CCD camera. For each slice, 30,000 points were scanned with 100 laser shots per position.The investigators encountered a variety of challenges during the study, especially with respect to synthesizing the tagged probes. They also faced sensitivity issues, which they addressed by developing a tagged single nucleotide that increased sensitivity by incorporating a tag for each T nucleotide basis.Ultimately, the experiments proved successful; the photocleavage induced by laser irradiation was efficient, yielding the characteristic signal that the researchers expected to see for the tag and showing that they could image a 180-kDa membrane protein and proenkephalin mRNA. They noted that, for oligonucleotides, uracil-tagged nucleotide probes provided the highest sensitivity and flexibility. The method revealed distributions of molecules on whole tissue sections with higher sensitivity than is available with most classical methods, and it imaged molecules that were otherwise inaccessible with Maldi techniques.Future applicationsThe technique could contribute to a variety of applications. “The concept is quite polyvalent, since it can be used for very different classes of molecules,” said Isabelle Fournier of Université des Sciences et Technologies de Lille. Potential clinical applications include early diagnosis of disease and treatment monitoring. In fact, the investigators have submitted a paper describing a study of ovarian cancer.“In this study, classical Maldi imaging was used for biomarker hunting,” Fournier said, “and the Tag-Mass concept was used for one part of the marker validation.” The method also can be used for basic science studies; for example, to obtain collocalization maps of various molecules within a specific area.The researchers are interested in testing other probes, such as lectins, to address sugars (glycoproteins, for example), or aptamers, to address peptides, proteins or drugs. They also are interested in testing isotopic tags for allowing relative quantification of two targeted molecules in the same sample. Finally, they plan to use tagged probes where the tag moiety is released by fragmentation in the Maldi ion source at the early stages of the desorption/ionization process.