Imaging mass spectrometry-based molecular histology differentiates microscopically identical and heterogeneous tumors.

Many tumors display significant cellular heterogeneity as well as molecular heterogeneity. Sensitive biomarkers that differentiate between diagnostically challenging tumors must contend with this heterogeneity. Mass spectrometry-based molecular histology of a patient series of heterogeneous, microscopically identical bone tumors highlighted the tumor cell types that could be characterized by a single profile and led to the identification of specific peptides that differentiate between the tumors.

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.

Differential recognition of vascular and parenchymal beta amyloid deposition.

By phage display, llama-derived heavy chain antibody fragments were selected from non-immune and immune libraries and tested for their affinity and specificity for beta amyloid by phage-ELISA, immunohistochemistry and surface plasmon resonance. We identified eight distinct heavy chain antibody fragments specific for beta amyloid. While three of them recognized vascular and parenchymal beta amyloid deposits, the remaining five heavy chain antibody fragments recognized vascular beta amyloid specifically, failing to bind to parenchymal beta amyloid. These heavy chain antibody fragments, selected from different libraries, demonstrated differential affinity for different epitopes when used for immunohistochemistry. These observations indicate that the llama heavy chain antibody fragments are the first immunologic probes with the ability to differentiate between parenchymal and vascular beta amyloid aggregates. This indicates that vascular and parenchymal beta amyloid deposits are heterogeneous in epitope presence/availability. The properties of these heavy chain antibody fragments make them potential candidates for use in in vivo differential diagnosis of Alzheimer disease and cerebral amyloid angiopathy. Continued use and characterization of these reagents will be necessary to fully understand the performance of these immunoreagents.

Imaging mass spectrometry data reduction: automated feature identification and extraction.

Imaging MS now enables the parallel analysis of hundreds of biomolecules, spanning multiple molecular classes, which allows tissues to be described by their molecular content and distribution. When combined with advanced data analysis routines, tissues can be analyzed and classified based solely on their molecular content. Such molecular histology techniques have been used to distinguish regions with differential molecular signatures that could not be distinguished using established histologic tools. However, its potential to provide an independent, complementary analysis of clinical tissues has been limited by the very large file sizes and large number of discrete variables associated with imaging MS experiments. Here we demonstrate data reduction tools, based on automated feature identification and extraction, for peptide, protein, and lipid imaging MS, using multiple imaging MS technologies, that reduce data loads and the number of variables by >100×, and that highlight highly-localized features that can be missed using standard data analysis strategies. It is then demonstrated how these capabilities enable multivariate analysis on large imaging MS datasets spanning multiple tissues.

MALDI imaging and profiling MS of higher mass proteins from tissue.

MALDI imaging and profiling mass spectrometry of proteins typically leads to the detection of a large number of peptides and small proteins but is much less successful for larger proteins: most ion signals correspond to proteins of m/z < 25,000. This is a severe limitation as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights exceeding 25 kDa. The detector technology typically used for protein imaging, a microchannel plate, is not well suited to the detection of high m/z ions and is prone to detector saturation when analyzing complex mixtures. Here we report increased sensitivity for higher mass proteins by using the CovalX high mass HM1 detector (Zurich, Switzerland), which has been specifically designed for the detection of high mass ions and which is much less prone to detector saturation. The results demonstrate that a range of different sample preparation strategies enable higher mass proteins to be analyzed if the detector technology maintains high detection efficiency throughout the mass range. The detector enables proteins up to 70 kDa to be imaged, and proteins up to 110 kDa to be detected, directly from tissue, and indicates new directions by which the mass range amenable to MALDI imaging MS and MALDI profiling MS may be extended.

Imaging mass spectrometry of myxoid sarcomas identifies proteins and lipids specific to tumour type and grade, and reveals biochemical intratumour heterogeneity.

Myxofibrosarcoma and myxoid liposarcomas are relatively common soft tissue tumours that are characterized by their so-called myxoid extracellular matrix and have to some extent overlap in histology. The exact composition and potential role of their myxoid extracellular matrix are insufficiently understood. To gain more insight into the biomolecular content of these tumours, we have studied 40 well-documented myxofibrosarcoma and myxoid liposarcoma cases using imaging mass spectrometry. This technique provides a multiplex biomolecular imaging analysis of the tissue, spanning multiple molecular domains and without a priori knowledge of the tissue's biomolecular content. We have developed experimental protocols for analysing the peptide, protein, and lipid content of myxofibrosarcoma and myxoid liposarcomas, and have detected proteins and lipids that are tumour-type and tumour-grade specific. In particular, lipid changes observed in myxoid liposarcomas could be related to pathways known to be affected during tumour progression. Unsupervised clustering of the biomolecular signatures was able to classify myxofibrosarcoma and myxoid liposarcomas according to tumour type and tumour grade. Closer examination of histologically similar regions in the tissues revealed intratumour heterogeneity, which was a consistent feature in each of the myxofibrosarcomas studied. In intermediate-grade myxofibrosarcoma, it was found that single tissue sections could contain regions with biomolecular profiles similar to high-grade and low-grade tumours, and that these regions were associated with the tumour's nodular structure, thus supporting a concept of tumour progression through clonal selection.

Calpain 3 is a rapid-action, unidirectional proteolytic switch central to muscle remodeling.

Calpain 3 (CAPN3) is a cysteine protease that when mutated causes Limb Girdle Muscular Dystrophy 2A. It is thereby the only described Calpain family member that genetically causes a disease. Due to its inherent instability little is known of its substrates or its mechanism of activity and pathogenicity. In this investigation we define a primary sequence motif underlying CAPN3 substrate cleavage. This motif can transform non-related proteins into substrates, and identifies >300 new putative CAPN3 targets. Bioinformatic analyses of these targets demonstrate a critical role in muscle cytoskeletal remodeling and identify novel CAPN3 functions. Among the new CAPN3 substrates are three E3 SUMO ligases of the Protein Inhibitor of Activated Stats (PIAS) family. CAPN3 can cleave PIAS proteins and negatively regulates PIAS3 sumoylase activity. Consequently, SUMO2 is deregulated in patient muscle tissue. Our study thus uncovers unexpected crosstalk between CAPN3 proteolysis and protein sumoylation, with strong implications for muscle remodeling.

MALDI mass spectrometry imaging of proteins exceeding 30,000 daltons.

BACKGROUND: Since its introduction 10 years ago by Caprioli and associates, MALDI mass spectrometry imaging has enabled spatial analysis of drugs, lipids, peptides, and polypeptides. In polypeptides, the detectable mass range is limited to small proteins with a mass less than 25 kDa. This is a limitation, as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights, exceeding 25 kDa. In the present work, we report the development of a novel strategy to observe higher mass proteins up to 30 kDa. MATERIAL/METHODS: We investigated the development of sample preparation methods based on hexafluoroisopropanol (1,1,1,3,3,3-hexaluoro-2-propanol) and 2,2,2-trifluoroethanol solvents for protein solubilization optimized for high-mass proteins. RESULTS: We were, for the first time in mass spectrometry imaging, able to detect to proteins up to 70 kDa directly from tissue. These developments indicate future avenues by which the sensitivity of protein mass spectrometry imaging can be further improved. We applied these developments to ovarian cancer and demonstrate that protein are similar to that which can be obtained using 2D gel based analyses. CONCLUSIONS: Increasing the possibility of detecting proteins and high-mass proteins is key for developing direct tissue proteomics and especially any potential functional investigation. These data will open the door of a novel step in mass spectrometry imaging.

Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry.

Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.

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. MALDI imaging MS can be directly applied to patient samples where its utility is to accomplish untargeted multiplex analysis of the tissue's protein content, enabling the different regions of the tissue to be differentiated on the basis of previously unknown protein profiles/biomarkers. The technique continues to rapidly develop and is now approaching the cusp whereby its potential to provide new diagnostic/prognostic tools for cancer patients can be routinely investigated. Here the latest methodological developments are summarized and its application to a range of tumors is reported in detail. The prospects of MALDI imaging MS are then described from the perspectives of modern pathological practice and MS-based proteomics, to ensure the outlook addresses real clinical needs and reflects the real capabilities of MS-based proteomics of complex tissue samples.

Automated imaging MS: Toward high throughput imaging mass spectrometry.

The term molecular histology has been used to convey the potential of imaging mass spectrometry to describe tissue by its constituent peptides and proteins, and to link this with established histological features. The low throughput of imaging mass spectrometry has been one of the factors inhibiting a full investigation of the clinical potential of molecular histology. Here we report the development of an automated set-up, consisting of a controlled environment sample storage chamber, a sample loading robot, and a MALDI-TOF/TOF mass spectrometer, all controlled by a single user interface. The automated set-up is demonstrated to have the positional stability and experimental reproducibility necessary for its clinical application.

Mass spectrometry image correlation: quantifying colocalization.

A typical imaging mass spectrometry data set can contain 100+ images, each describing the distribution of a specific biomolecule. Multivariate and hierarchical clustering techniques have been developed to investigate the correlations within a data set, and have revealed the differential patterns associated with different organs/anatomical features. These methods do not quantify the correlations between the hundreds of molecular distributions produced in an imaging mass spectrometry experiment, and are extremely difficult to apply to multiple tissue section investigations. This latter aspect includes quantifying the correlation between the results of repeat imaging mass spectrometry experiments, a crucial aspect for determining the significance of any measured changes in distribution. To date, the large chemical background and pixel-to-pixel variation in the images has limited the quantification of correlation between imaging mass spectrometry results. Here, we demonstrate how to quantify the correlations between imaging mass spectrometry images, both within a data set and between data sets.

Calpain 3 is a modulator of the dysferlin protein complex in skeletal muscle.

Muscular dystrophies comprise a genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and weakness. Two forms of limb-girdle muscular dystrophy, 2A and 2B, are caused by mutations in calpain 3 (CAPN3) and dysferlin (DYSF), respectively. While CAPN3 may be involved in sarcomere remodeling, DYSF is proposed to play a role in membrane repair. The coexistence of CAPN3 and AHNAK, a protein involved in subsarcolemmal cytoarchitecture and membrane repair, in the dysferlin protein complex and the presence of proteolytic cleavage fragments of AHNAK in skeletal muscle led us to investigate whether AHNAK can act as substrate for CAPN3. We here demonstrate that AHNAK is cleaved by CAPN3 and show that AHNAK is lost in cells expressing active CAPN3. Conversely, AHNAK accumulates when calpain 3 is defective in skeletal muscle of calpainopathy patients. Moreover, we demonstrate that AHNAK fragments cleaved by CAPN3 have lost their affinity for dysferlin. Thus, our findings suggest interconnectivity between both diseases by revealing a novel physiological role for CAPN3 in regulating the dysferlin protein complex.

AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration.

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair.

Monomeric and polymeric IgA show a similar association with the myeloid FcalphaRI/CD89.

IgA is found in both mucosal secretions and serum and is the dominant immunoglobulin isotype produced in humans. It exists in different molecular forms, namely monomeric IgA, dimeric IgA, polymeric IgA and secretory IgA, all exhibiting interactions with FcalphaRI/CD89 to some extent. CD89 is an activating, gamma-chain associated, Fc receptor for IgA expressed on myeloid cells. Here, we investigated the interaction of monomeric and polymeric IgA purified from human serum with CD89 using surface plasmon resonance. The results demonstrate a similar association for monomeric and polymeric IgA with CD89. In contrast, monomeric IgA dissociated more rapidly from CD89 than polymeric IgA. Removal of N-glycans from mIgA resulted is an increased association with CD89, whereas the dissociation was more rapid, resulting in binding comparable to that of untreated monomeric IgA. We conclude that the initial interaction of monomeric and polymeric IgA with CD89 is similar, whereas monomeric IgA dissociates more rapidly from CD89. In view of the large excess of monomeric IgA in serum, monomeric IgA will compete for CD89 interaction with polymeric IgA, thereby preventing cell activation initiated by receptor aggregation contributing to the anti-inflammatory role of IgA.

Generation of heavy-chain-only antibodies in mice.

We have generated transgenic mice containing hybrid llama/human antibody loci that contain two llama variable regions and the human D, J, and Cmu and/or Cgamma constant regions. Such loci rearrange productively and rescue B cell development efficiently without LC rearrangement. Heavy-chain-only antibodies (HCAb) are expressed at high levels, provided that the CH1 domain is deleted from the constant regions. HCAb production does not require an IgM stage for effective pre-B cell signaling. Antigen-specific heavy-chain-only IgM or IgGs are produced upon immunization. The IgG is dimeric, whereas IgM is multimeric. The chimeric HCAb loci are subject to allelic exclusion, but several copies of the transgenic locus can be rearranged and expressed successfully on the same allele in the same cell. Such cells are not subject to negative selection. The mice produce a full antibody repertoire and provide a previously undescribed avenue to produce specific human HCAb in the future.

Aberrant receptor-mediated endocytosis of Schistosoma mansoni glycoproteins on host lipoproteins.

BACKGROUND: Bilharzia is one of the major parasitic infections affecting the public health and socioeconomic circumstances in (sub) tropical areas. Its causative agents are schistosomes. Since these worms remain in their host for decades, they have developed mechanisms to evade or resist the immune system. Like several other parasites, their surface membranes are coated with a protective layer of glycoproteins that are anchored by a lipid modification. METHODS AND FINDINGS: We studied the release of glycosyl-phosphatidylinositol (GPI)-anchored proteins of S. mansoni and found them in the circulation associated with host lipoprotein particles. Host cells endocytosed schistosomal GPI-anchored proteins via their lipoprotein receptor pathway, resulting in disturbed lysosome morphology. In patients suffering from chronic schistosomiasis, antibodies attacked the parasite GPI-anchored glycoproteins that were associated with the patients' own lipoprotein particles. These immunocomplexes were endocytosed by cells carrying an immunoglobulin-Fc receptor, leading to clearance of lipoproteins by the immune system. As a consequence, neutral lipids accumulated in neutrophils of infected hamsters and in human neutrophils incubated with patient serum, and this accumulation was associated with apoptosis and reduced neutrophil viability. Also, Trypanosoma brucei, the parasite that causes sleeping sickness, released its major GPI-anchored glycoprotein VSG221 on lipoprotein particles, demonstrating that this process is generalizable to other pathogens/parasites. CONCLUSIONS: Transfer of parasite antigens to host cells via host lipoproteins disrupts lipid homeostasis in immune cells, promotes neutrophil apoptosis, may result in aberrant antigen presentation in host cells, and thus cause an inefficient immune response against the pathogen.

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants.

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.

Synthesis and antibody-binding studies of a series of parasite fuco-oligosaccharides.

Complex multifucosylated oligosaccharides are structural elements of glycoprotein and glycolipid subsets of larval, egg, and adult stages of Schistosoma, the parasitic worms that cause schistosomiasis, a serious disease affecting more than 200 million people in the tropics. The fucosylated structures are thought to play an important role in the immunology of schistosomiasis. Defined schistosomal oligosaccharides that enable immunological studies are difficult to obtain from natural sources. Therefore, we have chemically synthesized spacer-linked GlcNAc, Fucalpha1-3GlcNAc, Fucalpha1-2Fucalpha1-3GlcNAc, and Fucalpha1-2Fucalpha1-2Fucalpha1-3GlcNAc. This series of linear oligosaccharides was used to screen a library of anti-schistosome monoclonal antibodies by surface plasmon resonance spectroscopy. Interestingly, the reactive antibodies could be grouped according to their specificity for the different oligosaccharides tested, showing that these oligosaccharides form different immunological entities based on the number and linkage of the fucose residues. Subsequently, the thus defined monoclonal antibodies were used to visualize the expression of the corresponding oligosaccharide epitopes by adult Schistosoma mansoni worms.

Injection of recombinant FcalphaRI/CD89 in mice does not induce mesangial IgA deposition.

BACKGROUND: Earlier studies have suggested that complexes of the human IgA receptor FcalphaRI/CD89 with mouse IgA are pathogenic upon deposition in the renal mesangium. Transgenic mice expressing FcalphaRI/CD89 on macrophages/monocytes developed massive mesangial IgA deposition and a clinical picture of IgA nephropathy (IgAN). Based on these findings, the purpose of this study was to design an experimental model of IgAN by injection of human CD89 in mice. The interaction of mouse IgA with CD89 was investigated further. METHODS: Recombinant human soluble CD89 and a chimeric CD89-Fc protein were generated, produced, purified and injected in mice. Renal cryosections were stained for IgA and CD89. The interaction of mouse IgA with CD89 was analysed by fluorescence-activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA) and plasmon resonance technology. RESULTS: Injection of recombinant human CD89 did not result in significant IgA or CD89 deposition in the renal mesangium. However, CD89 staining in the liver was found to be positive. CD89 was rapidly cleared from circulation without signs of complex formation with IgA. FACS analysis, ELISA and plasmon resonance techniques all revealed a dose-dependent binding of human IgA to recombinant CD89, while no detectable binding was seen of mouse IgA, either of serum IgA or of different monoclonal mouse IgA preparations. CONCLUSIONS: An experimental model for IgAN in mice could not be obtained by injection of recombinant CD89. This is compatible with our in vitro biochemical data showing a lack of binding between recombinant human CD89 and mouse IgA.