Hospital libraries can provide an important source of tissue samples with formalin-fixed, paraffin-embedded tissues. Formalin fixation, typically followed by embedding in paraffin, can conserve and stabilize tissue samples for a number years and is a standard processing method used by pathologists. However, analyzing these samples has proved challenging in proteomics biomarker studies.Researchers have described two methods that enable direct analysis of formalin-fixed, paraffin-embedded tissue samples by Maldi,regardless of the conservation time — previously, analysis of older samples proved tremendously challenging. The methods rely on in situ enzymatic digestion to identify and even image peptides and proteins in the samples (LC = liquid chromatography, NanoESI/IT = nanoelectrospray ionization/ion trap, MS/MS = tandem mass spectrometry).Since the mid-1980s, researchers have established matrix-assisted laser-desorption/ionization (Maldi) mass spectrometry as a powerful method for biological studies. In the past 10 years, investigators have reported studies using the technique to localize and detect a range of compounds directly from tissue sections. The technique has been applied successfully only in fresh-frozen samples, though; tissues that have been stored for longer periods remain relatively inaccessible by Maldi mass spectrometry.In the April issue of the Journal of Proteome Research, researchers from Université des Sciences et Technologies de Lille in Villeneuve d’Ascq, France, from Université Pierre et Marie Curie in Paris, and from Université de Sherbrooke in Quebec, Canada, described two methods applicable to direct analysis of formalin-fixed, paraffin-embedded tissue samples by Maldi mass spectrometry — one that can be used with tissue samples stored up to one year and one that can be used with all samples no matter the conservation time.Using the methods, researchers acquired Maldi molecular images from 2-year-oldformalin-fixed, paraffin-embedded (FFPE) rat brain tissue sections. A picture and the morphology of the rat brain, in the two bottom right panels, are provided for comparison. Reprinted with permission of the Journal of Proteome Research.Direct analysis of formalin-fixed, paraffin-embedded tissue samples is complicated by the fact that the process induces cross-linking of proteins. Cross-linking continues over time, further confounding analysis of older samples. Direct analysis is still possible in samples less than 1 year old, albeit with considerable loss of resolution and signal, particularly with higher-mass proteins. The investigators reported a solution to this loss that uses a different Maldi matrix (2,4-DNPH). Use of the matrix enabled them to measure peptides with a normal resolution, and it even allowed direct Maldi imaging of tissue with results comparable to those obtained from frozen conserved samples.Another Maldi matrixWith samples older than a year, however, the extent of cross-linking is too high to obtain good signal. “In these cases, the strategy was to use in situ enzymatic digestion” after paraffin removal, explained researcher Isabelle Fournier. “This has proved to work very well with such samples.” The strategy enabled Maldi imaging also by reconstructing images on the basis of the digested peptides. Fournier noted, however, that the enzyme must be microspotted to avoid delocalization of the peptides, and the matrix must be deposited on the same raster before the Maldi scan.The investigators demonstrated the technique by performing Maldi mass spectrometry and Maldi imaging on tissue samples from rat brains stored for either six months or two years. They performed Maldi time-of-flight mass spectroscopy measurements using a mass spectrometer with a 337-nm pulsed nitrogen laser operating at 3 Hz and with a 2-ns pulse width made by Applied Biosystems of Framingham, Mass. They used an Ultraflex II Tof-Tof made by Bruker Daltonics of Bremen, Germany, to perform Maldi imaging of tissue samples stored for six months as well as to perform Maldi mass spectrometry experiments on 2-year-old samples after in situ digestion of the whole tissue section. Analysis was performed on an ion trap mass spectrometer made by Thermo Fisher Scientific of Waltham, Mass.The experiments confirmed the efficacy of the techniques and underscored their potential for a variety of applications. By providing the molecular distribution of peptides and proteins in formalin-fixed and paraffin-embedded tissues, they could contribute to fundamental biology studies as well as to clinical applications.The researchers will continue to develop the Maldi mass spectrometry techniques — to be able to identify proteins directly from the tissue using de novo sequencing by means of mass spectrometry, for instance. “In this strategy,” Fournier noted, “we are optimizing N-terminal derivation of proteins that allow for increasing fragmentation yield and that orient fragmentation to facilitate data interpretations and primary structure determination.”They also are exploring different applications of the techniques, such as the study of Parkinson’s disease. They have been able to identify several proteins in 9-year-old formalin-fixed, paraffin-embedded tissue samples that are modulated in rat tissue in which Parkinson’s disease was induced, with respect to control samples. “Some of the proteins have already been found using classical proteomics or genomics approaches,” Fournier said, “but a few sets of them are new. All of these results should be published soon.”