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.     

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.     

Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometry.

We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 microL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.     

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.     

Tissue profiling by mass spectrometry: a review of methodology and applications

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has become a valuable tool to address a broad range of questions in many areas of biomedical research. One such application allows spectra to be obtained directly from intact tissues, termed "profiling" (low resolution) and "imaging" (high resolution). In light of the fact that MALDI tissue profiling allows over a thousand peptides and proteins to be rapidly detected from a variety of tissues, its application to disease processes is of special interest. For example, protein profiles from tumors may allow accurate prediction of tumor behavior, diagnosis, and prognosis and uncover etiologies underlying idiopathic diseases. MALDI MS, in conjunction with laser capture microdissection, is able to produce protein expression profiles from a relatively small number of cells from specific regions of heterogeneous tissue architectures. Imaging mass spectrometry enables the investigator to assess the spatial distribution of proteins, drugs, and their metabolites in intact tissues. This article provides an overview of several tissue profiling and imaging applications performed by MALDI MS, including sample preparation, matrix selection and application, histological staining prior to MALDI analysis, tissue profiling, imaging, and data analysis. Several applications represent direct translation of this technology to clinically relevant problems.     

Small protein biomarkers of culture in Bacillus spores detected using capillary liquid chromatography coupled with matrix assisted laser desorption/ionization mass spectrometry

Capillary liquid chromatography (cLC) coupled with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) was used to compare small proteins and peptides extracted from Bacillus subtilis spores grown on four different media. A single, efficient protein separation, compatible with MALDI-MS analysis, was employed to reduce competitive ionization between proteins, and thus interrogate more proteins than possible using direct MALDI-MS. The MALDI-MS data files for each fraction are assembled as two-dimensional data sets of retention time and mass information. This method of visualizing small protein data required careful attention to background correction as well as mass and retention time variability. The resulting data sets were used to create comparative displays of differences in protein profiles between different spore preparations. Protein differences were found between two different solid media in both phase bright and phase dark spore phenotype. The protein differences between two different liquid media were also examined. As an extension of this method, we have demonstrated that candidate protein biomarkers can be trypsin digested to provide identifying peptide fragment information following the cLC-MALDI experiment. We have demonstrated this method on two markers and utilized acid breakdown information to identify one additional marker for this organism. The resulting method can be used to identify discriminating proteins as potential biomarkers of growth media, which might ultimately be used for source attribution.     

Strain-based sequence variations and structure analysis of murine prostate specific spermine binding protein using mass spectrometry

Mouse spermine binding protein (SBP) has been characterized using mass spectrometry, including its localization within the prostate, sequence verification, and its posttranslational modifications. MALDI (matrix-assisted laser desorption/ionization) mass spectrometry was employed for localization of proteins expressed by different lobes of the mouse prostate obtained after tissue blotting on a polyethylene membrane. The mass spectra showed complex protein profiles that were different for each lobe of the prostate. The prostate-specific spermine binding protein (SBP), primarily identified by its in-source decay fragment ion signals, was found predominantly expressed by the ventral lobe of the prostate. The MALDI in-source decay measurements combined with nanoESI (nanoelectrospay ionization) MS/MS measurements obtained after specific proteolysis of SBP, allowed the exact positioning of a single N-linked carbohydrate group, and the identification of a pyroglutamate residue at the sequence N-terminus. The N-linked carbohydrate component was further investigated and the general pattern of the N-linked carbohydrate identified. The presence of a disulfide bridge between cysteine78 and cysteine124 was also established. The full sequence characterization of SBP showed several strain-based sequence differences when compared to the published gene sequence.     

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.     

Integrating spatially resolved three-dimensional MALDI IMS with in vivo magnetic resonance imaging

We have developed a method for integrating three dimensional-volume reconstructions of spatially resolved matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) ion images of whole mouse heads with high-resolution images from other modalities in an animal-specific manner. This approach enabled us to analyze proteomic profiles from MALDI IMS data with corresponding in vivo data provided by magnetic resonance imaging.     

Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system

Seminal plasma is a fluid that originates from the testis, epididymis,prostate, and seminal vesicles, and hence, proteomic studies may identify potential markers of infertility and other diseases of the genito-urinary tract. We profiled the proteomes of pooled seminal plasma from fertile Control and post-vasectomy (PV) men. PV seminal plasma samples are void of proteins originating from the testis and the epididymis due to ligation of the vas deferens, and hence, comparative analysis of Control and PV data sets allows for identification of proteins originating from these tissues. Utilizing offline MudPIT and high-resolution mass spectrometry, we were able to identify over 2000 proteins in Control and PV pools each and over 2300 proteins all together. With semiquantitative analysis using spectral counting, we catalogued 32 proteins unique to Control, 49 at lower abundance in PV, 3 unique to PV, and 25 at higher abundance in PV. We believe that proteins unique to Control or at lower abundance in PV have their origin in the testis and the epididymis. Public databases have confirmed that many of these proteins originate from the testis and epididymis and are linked to the reproductive tract. These proteins may serve as candidate biomarkers for future studies of infertility and urogenital diseases.     

MALDI imaging of post‐mortem human spinal cord in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressing disease of the central nervous system that is characterized by motor neuron degeneration in the brainstem and the spinal cord. Matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry is an emerging powerful technique that allows for spatially resolved, comprehensive, and specific characterization of molecular species in situ. In this study, we report for the first time the MALDI imaging‐based spatial protein profiling and relative quantification of post‐mortem human spinal cord samples obtained from ALS patients and controls. In normal spinal cord, protein distribution patterns were well in line with histological features. For example, thymosin beta 4, ubiquitin, histone proteins, acyl‐CoA‐binding protein, and macrophage inhibitory factor were predominantly localized to the gray matter. Furthermore, unsupervised statistics revealed a significant reduction of two protein species in ALS gray matter. One of these proteins (m/z 8451) corresponds to an endogenous truncated form of ubiquitin (Ubc 1–76), with both C‐terminal glycine residues removed (Ubc‐T/Ubc 1–74). This region‐specific ubiquitin processing suggests a disease‐related change in protease activity. These results highlight the importance of MALDI mass spectrometry as a versatile approach to elucidate molecular mechanisms of neurodegenerative diseases.     

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.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.     

Differential proteomic analysis of the mouse retina: the induction of crystallin proteins by retinal degeneration in the rd1 mouse

We have applied proteomic analysis to the degeneration of photoreceptors. In the rd1 mouse, a recessive mutation in the PDE6B gene leads to rapid loss of rods through apoptosis. By 5 wk postnatal, virtually all rod photoreceptors have degenerated, leaving one row of cones that degenerates secondarily. In order to assess comparative protein expression, proteins extracted from whole retina were resolved on a two-dimensional gel and identified by mass spectrometry combined with database screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry coupled to peptide mass fingerprinting was sufficient to identify most of the proteins, the remaining being identified with additional sequence information obtained by nano-electrospray ionization tandem mass spectrometry or liquid chromatography tandem mass spectrometry. The study revealed 212 spots, grouped into 109 different proteins. Differential analysis showed loss of proteins involved in the rod-specific phototransduction cascade, as well as induction of proteins from the crystallin family, in response to retinal degeneration. Identification of such pathways may contribute to new therapeutic approaches.     

Identification of Protein Markers Specific for Papillary Renal Cell Carcinoma Using Imaging Mass Spectrometry

Since the emergence of proteomics methods, many proteins specific for renal cell carcinoma (RCC) have been identified. Despite their usefulness for the specific diagnosis of RCC, such proteins do not provide spatial information on the diseased tissue. Therefore, the identification of cancer-specific proteins that include information on their specific location is needed. Recently, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) based imaging mass spectrometry (IMS) has emerged as a new tool for the analysis of spatial distribution as well as identification of either proteins or small molecules in tissues. In this report, surgical tissue sections of papillary RCC were analyzed using MALDI-IMS. Statistical analysis revealed several discriminative cancer-specific m/z-species between normal and diseased tissues. Among these m/z-species, two particular proteins, S100A11 and ferritin light chain, which are specific for papillary RCC cancer regions, were successfully identified using LC-MS/MS following protein extraction from independent RCC samples. The expressions of S100A11 and ferritin light chain were further validated by immunohistochemistry of human tissues and tissue microarrays (TMAs) of RCC. In conclusion, MALDI-IMS followed by LC-MS/MS analysis in human tissue identified that S100A11 and ferritin light chain are differentially expressed proteins in papillary RCC cancer regions.     

Direct profiling and imaging of plant metabolites in intact tissues by using colloidal graphite-assisted laser desorption ionization mass spectrometry

Laser desorption/ionization (LDI)-based imaging mass spectrometry (MS) has been applied to several biological systems to obtain information about both the identities of the major chemical species and their localization. Colloidal graphite-assisted LDI (GALDI) MS imaging was introduced for the imaging of small molecules such as phospholipids, cerebrosides, oligosaccharides, flavonoids, and other secondary metabolites with high spatial homogeneity due to finely dispersed particles. Mass profiles and images of Arabidopsis thaliana have been recorded directly from various plant surfaces and cross sections. The main targeted metabolites were flavonoids and cuticular waxes, both of which are important in many aspects of functional genomics, proteomics, and metabolomics. The mass spectral profiles revealed tissue-specific accumulation of flavonoids in flowers and petals. In addition, many other location-specific ions were observed. The location and the degree of light-induced accumulation of flavonoids in stem sections were successfully probed by GALDI MS.     

Mass spectrometry imaging is moving toward drug protein co-localization

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10-20μm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies.     

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.