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.     

Peptide and protein imaging mass spectrometry in cancer research

MALDI mass spectrometry is able to acquire protein profiles directly from tissue that can describe the levels of hundreds of distinct proteins. MALDI imaging MS can simultaneously reveal how each of these proteins varies in heterogeneous tissues. Numerous studies have now demonstrated how MALDI imaging MS can generate different protein profiles from the different cell types in a tumor, which can act as biomarker profiles or enable specific candidate protein biomarkers to be identified.     

New access to Alzheimer’s and other neurodegenerative diseases

Single-cell analysis is gaining popularity in the field of mass spectrometry as a method for analyzing protein and peptide content in cells. The spatial resolution of MALDI mass spectrometry (MS) imaging is by a large extent limited by the laser focal diameter and the displacement of analytes during matrix deposition. Owing to recent advancements in both laser optics and matrix deposition methods, spatial resolution on the order of a single eukaryotic cell is now achievable by MALDI MS imaging. Provided adequate instrument sensitivity, a lateral resolution of ?10 µm is currently attainable with commercial instruments. As a result of these advances, MALDI MS imaging is poised to become a transformative clinical technology. In this article, the crucial steps needed to obtain single-cell resolution are discussed, as well as potential applications to disease research.     

Recent advances in single-cell MALDI mass spectrometry imaging and potential clinical impact

Single-cell analysis is gaining popularity in the field of mass spectrometry as a method for analyzing protein and peptide content in cells. The spatial resolution of MALDI mass spectrometry (MS) imaging is by a large extent limited by the laser focal diameter and the displacement of analytes during matrix deposition. Owing to recent advancements in both laser optics and matrix deposition methods, spatial resolution on the order of a single eukaryotic cell is now achievable by MALDI MS imaging. Provided adequate instrument sensitivity, a lateral resolution of approximately 10 μm is currently attainable with commercial instruments. As a result of these advances, MALDI MS imaging is poised to become a transformative clinical technology. In this article, the crucial steps needed to obtain single-cell resolution are discussed, as well as potential applications to disease research.     

Assessing protein patterns in disease using imaging mass spectrometry

Direct tissue profiling and imaging mass spectrometry (MS) provides a detailed assessment of the complex protein pattern within a tissue sample. MALDI MS analysis of thin tissue sections results in over of 500 individual protein signals in the mass range of 2 to 70 kDa that directly correlate with protein composition within a specific region of the tissue sample. To date, profiling and imaging MS has been applied to multiple diseased tissues, including human gliomas and nonsmall cell lung cancer. Interrogation of the resulting complex MS data sets has resulted in identification of both disease-state and patient-prognosis specific protein patterns. These results suggest the future usefulness of proteomic information in assessing disease progression, prognosis, and drug efficacy.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry: a promising technique for reproductive research

Matrix-assisted laser desorption/ionization (MALDI) tissue imaging mass spectrometry is particularly promising among the numerous applications of mass spectrometry. It is used for probing and analyzing the spatial arrangement of a wide range of molecules, including proteins, peptides, lipids, drugs, and metabolites, directly in thin slices of tissue. In the field of proteomics, the technology avoids tedious and time-consuming extraction and fractionation steps classically required for sample analysis. MALDI imaging mass spectrometry is increasingly recognized as a powerful method for clinical proteomics, particularly in cancer research. The technology has particular potential for the discovery of new tissue biomarker candidates, classification of tumors, early diagnosis or prognosis, elucidating pathogenesis pathways, and therapy monitoring. Over recent years, MALDI imaging mass spectrometry has been used for molecular profiling and imaging directly in male and female reproductive tissues. This review will consider some of the recent publications in the field, addressing a range of issues covering embryo development, gene expression product profiling during gametogenesis, and seeking and identifying biomarkers of reproductive cancers. The wealth of advances in mass spectrometry imaging will inevitably attract biologists and clinicians as the advantages and power of this technology become more widely known. This review will also discuss bottlenecks and the many technical issues that remain to be resolved before laboratories in the field can adopt the technology. We foresee that MALDI imaging mass spectrometry will have a major impact in reproductive research by opening new avenues to the understanding of various molecular mechanisms and the diagnosis of reproductive pathologies.     

Protein expression profiling of endometrial malignancies reveals a new tumor marker: chaperonin 10

Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that of breast, lung, and colorectal cancers. At present, no serum tumor markers are available for the monitoring of endometrial carcinoma patients, and patients with recurrent disease are detected only following the development of symptoms or abnormalities in imaging assessments. Similarly, no screening tools are available for endometrial carcinoma. Protein profiling by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a sensitive and fast method of analysis for small proteins or peptides to yield specific biomarkers. In this study, a variety of normal and malignant endometrial tissue samples were fractionated and analyzed by SELDI-TOF MS (SELDI is a version of MALDI utilizing protein "chips"). A number of proteins displayed differential expression in malignant endometrial tissues. One of the prominent proteins fractionated by weak cation exchange chromatography and displaying enhanced expression in these malignant tissues was identified as chaperonin 10. The increased expression of chaperonin 10 in malignant endometrial tissues was further confirmed by parallel Western blot and immunohistochemistry analyses.     

Mass spectrometry imaging with high resolution in mass and space

Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of ‘mass spectrometry imaging with high resolution in mass and space.’ Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5–10 μm. Proteins were analyzed after on-tissue tryptic digestion at 50-μm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 μm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level.     

Mass spectrometry imaging and profiling of single cells

Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies-secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI MS)-are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enables new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.     

Combined experimental and statistical strategy for mass spectrometry based serum protein profiling for diagnosis of breast cancer: a case-control study

Serum protein profiling by mass spectrometry is a promising method for early detection of cancer. We have implemented a combined strategy based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and statistical data analysis for serum protein profiling and applied it in a well-described breast cancer case-control study. A rigorous sample collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum samples obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total of nine mass spectrometric protein profiles were obtained for each serum sample. A total of 533 common peaks were defined and represented a 'reference protein profile'. Among these 533 common peaks, we identified 72 peaks exhibiting statistically significant intensity differences ( p < 0.01) between cases and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss of sensitivity and specificity. We conclude that optimized serum sample handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis.     

Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

Multiple statistical analysis techniques corroborate intratumor heterogeneity in imaging mass spectrometry datasets of myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information.     

Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections

MALDI MS imaging mass spectrometry can be used to map the distribution of targeted compounds in tissue sections with a spatial resolution currently of about 50 microm, providing important molecular information in many areas of biological research. After matrix application, a raster of a section by the laser beam yields ions from compounds in a tissue mass-to-charge range from 1000 to over 100000. Two-dimensional intensity maps can then be reconstructed to provide specific molecular images of a tissue.     

Protein profiling by the combination of two independent mass spectrometry techniques

Protein profiling in the high-throughput mode is a most useful technique that allows formation of reference databases for cells and tissues and performance of comparative proteomics. In the proposed protocol protein extraction from tissues is followed by 2D gel electrophoresis (2DE) with subsequent in-gel digestion and identification of soluble proteins by two individual mass spectrometric techniques, tandem matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano-liquid chromatography (nano-LC)-MS/MS. The proposed combined use of these two MS approaches leads to a very high identification rate of well-separated protein spots from a gel. In the first step 2DE separates high-abundance proteins (those visualized by nonsensitive Coomassie blue staining) that are subsequently picked, digested and aliquoted for MS applications. Protein samples not identified by MALDI-MS or MS/MS (77% of all spots) are finally unambiguously identified by nano-LC-MS/MS (total identification rate 94%). This protocol can be completed in 6 weeks.     

MALDI mass spectrometry in prostate cancer biomarker discovery

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) is a highly versatile and sensitive analytical technique, which is known for its soft ionisation of biomolecules such as peptides and proteins. Generally, MALDI MS analysis requires little sample preparation, and in some cases like MS profiling it can be automated through the use of robotic liquid-handling systems. For more than a decade now, MALDI MS has been extensively utilised in the search for biomarkers that could aid clinicians in diagnosis, prognosis, and treatment decision making. This review examines the various MALDI-based MS techniques like MS imaging, MS profiling and proteomics in-depth analysis where MALDI MS follows fractionation and separation methods such as gel electrophoresis, and how these have contributed to prostate cancer biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.     

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.     

PAXgene fixation enables comprehensive metabolomic and proteomic analyses of tissue specimens by MALDI MSI

An alcohol-based non-crosslinking tissue fixative, PAXgene Tissue System, has been proposed as alternative fixation method to formalin, providing superior and morphological preservation. To date, metabolites have not been assessed in PAXgene-fixed tissues. The study focuses on a comparison between PAXgene and standard formalin fixation for metabolomic analysis by MALDI mass spectrometry imaging. Therefore, fifty-six samples from seven mice organs were fixed with PAXgene (PFPE) or formalin (FFPE), embedded in paraffin, and processed to a tissue microarray. PAXgene was able to spatially preserve metabolites in organs achieving an overlap of common metabolites ranging from 34 to 78% with FFPE. Highly similar signal intensities and visualization of molecules demonstrated negligible differences for metabolite imaging on PFPE compared to FFPE tissues. In addition, we performed proteomic analysis of intact proteins and peptides derived from enzymatic digestion. An overlap of 33 to 58% was found between FFPE and PFPE tissue samples in peptide analysis with a higher number of PFPE-specific peaks. Analysis of intact proteins achieved an overlap in the range of 0 to 28% owing to the poor detectability of cross-linked proteins in formalin-fixed tissues. Furthermore, metabolite and peptide profiles obtained from PFPE tissues were able to correctly classify organs independent of the fixation method, whereas a distinction of organs by protein profiles was only achieved by PAXgene fixation. Finally, we applied MALDI MSI to human biopsies by sequentially analyzing metabolites and peptides within the same tissue section. Concerning prospective studies, PAXgene can be used as an alternative fixative for multi-omic tissue analysis.     

Molecular histoproteomy by MALDI mass spectrometry imaging to uncover markers of the impact of Nosema on Apis mellifera

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology used to investigate the spatio-temporal distribution of a huge number of molecules throughout a body/tissue section. In this paper, we report the use of MALDI IMS to follow the molecular impact of an experimental infection of Apis mellifera with the microsporidia Nosema ceranae. We performed representative molecular mass fingerprints of selected tissues obtained by dissection. This was followed by MALDI IMS workflows optimization including specimen embedding and positioning as well as washing and matrix application. We recorded the local distribution of peptides/proteins within different tissues from experimentally infected versus non infected honeybees. As expected, a distinction in these molecular profiles between the two conditions was recorded from different anatomical sections of the gut tissue. More importantly, we observed differences in the molecular profiles in the brain, thoracic ganglia, hypopharyngeal glands, and hemolymph. We introduced MALDI IMS as an effective approach to monitor the impact of N. ceranae infection on A. mellifera. This opens perspectives for the discovery of molecular changes in peptides/proteins markers that could contribute to a better understanding of the impact of stressors and toxicity on different tissues of a bee in a single experiment.     

Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation,bottom‐up and top‐down mass spectrometry

Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed.