Molecular profiling of experimental Parkinson's disease: direct analysis of peptides and proteins on brain tissue sections by MALDI mass spectrometry

Direct molecular profiling of biological samples using matrix-assisted laser desorption ionization mass spectrometry is a powerful tool for identifying phenotypic markers. In this report, protein profiling was used for the first time to generate peptide and protein profiles of brain tissue sections obtained from experimental Parkinson's disease (unilaterally 6-hydroxydopamine treated rats). The mass spectrometer was used to map the peptide and protein expression directly on 12 microm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein expression profile differences were found in the dopamine depleted side of the brain when compared to the corresponding intact side, for example, calmodulin, cytochrome c, and cytochrome c oxidase. An increased ratio of post-translational modifications such as acetylations were found in the striatum of proteins in the dopamine depleted side of the brain. These modifications were decreased after subchronic administration of L-Dopa. The present study shows that unique protein profiles can be obtained in specific brain regions (and subregions) directly on brain tissue sections and allows for the study of complex biochemical processes such as those occurring in experimental Parkinson's disease.     

Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE-TMAs using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE-TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis.     

Molecular imaging of thin mammalian tissue sections by mass spectrometry

Imaging of tissue sections by mass spectrometry provides a detailed molecular picture containing information on both the abundance and distribution of many constituent compounds. Mass spectra are acquired directly from fresh frozen tissue sections using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS); sample preparation and data collection mode determine the spatial resolution or surface area of the section represented in each mass spectrum. Statistical analyses of the individual ion signatures yield biomarkers whose abundances correlate to cell development processes, tumorigenesis and/or drug treatment. In an alternate mode, the generation of intensity maps for individual ions provides a visual representation of the distribution of each species throughout the section at spatial resolutions as small as 50 microm. The availability of this molecular information is likely to be of great value to clinicians and should lead to improved therapeutic efficacy in the future.     

Exploring proteins in Anopheles gambiae male and female antennae through MALDI mass spectrometry profiling

MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.     

A simple desalting method for direct MALDI mass spectrometry profiling of tissue lipids

Direct MALDI-mass spectrometry (MALDI-MS) profiling of tissue lipids often observes isobaric phosphatidylcholine (PC) species caused by the endogenous alkali metal ions that bias the relative abundance of tissue lipids. Fresh rat brain cryosections were washed with 70% etha­nol (EtOH), water (H₂O), or 150 mM ammonium acetate (NH₄Ac), and the desalting effectiveness of each fluid was evaluated by MALDI-MS profiling of PC and sphingomyelin (SM) species in tissue and in the washing runoff. The results indicated that EtOH and H₂O only partially desalted the tissue lipids, yet both substantially displaced the tissue lipids to the washing runoffs. On the other hand, NH₄Ac effectively desalted the tissue lipids and produced a runoff containing no detectable PCs or SMs. NH₄Ac wash also unveiled the underlying changes of PCs and SMs in the infarcted rat cortex previously masked by edema-caused increase of tissue sodium. The MS/MS of an isobaric PC in the infarcted cortex revealed the precursor change as the result of NH₄Ac wash and confirmed the desalting effectiveness of such wash. Other than desalting, NH₄Ac wash also removes contaminants in tissue, enhances the overall spectral quality, and benefits additionally in profiling of biological molecules in tissue.     

MALDI imaging mass spectrometry of human tissue: method challenges and clinical perspectives

The molecular complexity of biological tissue and the spatial and temporal variation in the biological processes involved in human disease requires new technologies and new approaches to provide insight into disease processes. Imaging mass spectrometry is an effective tool that provides molecular images of tissues in the molecular discovery process. The analysis of human tissue presents special challenges and limitations because the heterogeneity among human tissues and diseases is much greater than that observed in animal models, and discoveries made in animal tissues might not translate well to their human counterparts. In this article, we briefly review the challenges of imaging human tissue using mass spectrometry and suggest approaches to address these issues.     

High-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue microarrays using MALDI imaging mass spectrometry

A novel method for high-throughput proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMA) is described using on-tissue tryptic digestion followed by MALDI imaging MS. A TMA section containing 112 needle core biopsies from lung-tumor patients was analyzed using MS and the data were correlated to a serial hematoxylin and eosin (H&E)-stained section having various histological regions marked, including cancer, non-cancer, and normal ones. By correlating each mass spectrum to a defined histological region, statistical classification models were generated that can sufficiently distinguish biopsies from adenocarcinoma from squamous cell carcinoma biopsies. These classification models were built using a training set of biopsies in the TMA and were then validated on the remaining biopsies. Peptide markers of interest were identified directly from the TMA section using MALDI MS/MS sequence analysis. The ability to detect and characterize tumor marker proteins for a large cohort of FFPE samples in a high-throughput approach will be of significant benefit not only to investigators studying tumor biology, but also to clinicians for diagnostic and prognostic purposes.     

MALDI imaging mass spectrometry for direct tissue analysis: technological advancements and recent applications

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a method that allows the investigation of the molecular content of tissues within its morphological context. Since it is able to measure the distribution of hundreds of analytes at once, while being label free, this method has great potential which has been increasingly recognized in the field of tissue-based research. In the last few years, MALDI-IMS has been successfully used for the molecular assessment of tissue samples mainly in biomedical research and also in other scientific fields. The present article will give an update on the application of MALDI-IMS in clinical and preclinical research. It will also give an overview of the multitude of technical advancements of this method in recent years. This includes developments in instrumentation, sample preparation, computational data analysis and protein identification. It will also highlight a number of emerging fields for application of MALDI-IMS like drug imaging where MALDI-IMS is used for studying the spatial distribution of drugs in tissues.     

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.     

Drug localization in different lung cancer phenotypes by MALDI mass spectrometry imaging

Lung cancer is a common cause of cancer mortality in the world, largely due to the risk factor of tobacco smoking. The drug therapy at the molecular level includes targeting the epidermal growth factor receptor (EGFR) tyrosine kinase activity by using inhibitors, such as erlotinib (Tarceva) and gefitinib (Iressa). The heterogeneity of disease phenotypes and the somatic mutations presented in patient populations have a great impact on the efficacy of treatments using targeted personalized medicine. In this study, we report on basic physical and chemical properties of erlotinib and gefitinib in three different lung cancer tumor phenotypes, using MALDI instrumentation in imaging mode, providing spatial localization of drugs without chemical labeling. Erlotinib and gefitinib were analyzed in i) planocellular lung carcinoma, ii) adenocarcinoma and iii) large cell lung carcinoma following their deposition on the tissue surfaces by piezo-dispensing, using a controlled procedure. The importance of high-resolution sampling was crucial in order to accurately localize the EGFR tyrosine kinase inhibitors deposited in heterogeneous cancer tissue compartments. This is the first report on personalized drug characterization with localizations at a lateral resolution of 30μm, which allowed us to map these compounds at attomolar concentrations within the lung tumor tissue microenvironments.     

Strain differentiation of Staphylococcus aureus by means of direct MALDI TOF mass spectrometry profiling

Staphylococcus aureus is one of the most interesting microbial species in clinical studies. It is characterized by a wide extent of strain diversity, first of all, due to variability in virulence and pathogenicity. The aim of this study was to test the method of rapid Staphylococcus strain differentiation by a certain sign based on registration of characteristics features of MALDI mass spectra accumulated during direct protein profiling of the bacterial cell. The model signs registered as strain differences included production of β-lactamase and α-hemolysin encoded by blaZ and hla genes, respectively. The mathematical analysis of MALDI mass spectra accumulated for 53 S. aureus isolates using the clustering genetic algorithm resulted in generation of two independent classification models, which could differentiate the strains by the considered features. Using statistical contribution of each mass peak to the model, the most significant peaks (masses), which could be considered as the markers of Staphylococcus strain differences, were found. The generated diagnostic models were characterized by the following sensitivity and specificity coefficients: 97.5 and 82.5%, respectively, for strain differentiation by β-lactamase production and 90.0 and 88.7% by the presence of α-hemolysin.     

Monitoring mouse prostate development by profiling and imaging mass spectrometry

Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1-5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15-week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially alpha-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer.     

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 μm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 μm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.     

MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples

Histone post-translational modifications (PTMs), histone variants and enzymes responsible for the incorporation or the removal of the PTMs are being increasingly associated with human disease. Combinations of histone PTMs and the specific incorporation of variants contribute to the establishment of cellular identity and hence are potential markers that could be exploited in disease diagnostics and prognostics and therapy response prediction. Due to the scarcity of suitable antibodies and the pre-requirement of tissue homogenization for more advanced analytical techniques, comprehensive information regarding the spatial distribution of these factors at the tissue level has been lacking. MALDI imaging mass spectrometry provides an ideal platform to measure histone PTMs and variants from tissues while maintaining the information about their spatial distribution. Discussed in this review are the relevance of histones in the context of human disease and the contribution of MALDI imaging mass spectrometry in measuring histones in situ.     

Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids

Lipids are a major component of cells and play a variety of roles in organisms. In general, they play a key role in the structural composition of membranes. Some lipids, such as sphingoglycolipids, however, are also mediators of different biological processes, including protein transport, regulation of cell growth, cellular morphogenesis, neuronal plasticity, and regulation of the immune response. With the advent of MALDI mass spectrometry imaging (MALDI MSI), lipids have begun to be intensively investigated by several groups. Here we present a novel development in the detection and study of lipids using an automatic microspotter coupled to specific liquid ionic matrixes based on a 2,5-DHB matrix (i.e., 2,5-DHB/ANI, 2,5-DHB/Pyr, and 2,5-DHB/3-AP). This development allows to decrease the time of the sample preparation by comparison with crystalline 2,5-DHB as matrix and was validated on human ovarian cancer biopsies to demonstrate its use as a precise procedure that is particularly useful for specific diagnoses.     

MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.     

MALDI imaging mass spectrometry: molecular snapshots of biochemical systems

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.     

Profiling and imaging proteins in the mouse epididymis by imaging mass spectrometry

Different aspects of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) have been used as discovery tools to obtain global and time-correlated information on the local proteomic composition of the sexually mature mouse epididymis from both qualitative and semiquantitative points of view. Tissue sections and laser captured microdissected cells and secretory products were analyzed by MALDI-MS and from the recovered protein profiles, over 400 different proteins were monitored. Over 50 of these, some of which have been identified, displayed regionalized behavior from caput to cauda within the epididymis. Combining the information obtained from high-resolution imaging mass spectrometry and laser captured microdissection experiments, numerous proteins were localized within the epididymis at the cellular level. Furthermore, from the signal intensities observed in the different protein profiles organized in space, semiquantitative information for each protein was obtained.