RESUMEN
Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC liver metastases (CRLM) are often resistant to conventional treatments, with high rates of recurrence. Therefore, it is crucial to identify biomarkers for CRLM patients that predict cancer progression. This study utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially map the CRLM tumour proteome. CRLM tissue microarrays (TMAs) of 84 patients were analysed using tryptic peptide MALDI-MSI to spatially monitor peptide abundances across CRLM tissues. Abundance of peptides was compared between tumour vs stroma, male vs female and across three groups of patients based on overall survival (0-3 years, 4-6 years, and 7+ years). Peptides were then characterised and matched using LC-MS/MS. A total of 471 potential peptides were identified by MALDI-MSI. Our results show that two unidentified m/z values (1589.876 and 1092.727) had significantly higher intensities in tumours compared to stroma. Ten m/z values were identified to have correlation with biological sex. Survival analysis identified three peptides (Histone H4, Haemoglobin subunit alpha, and Inosine-5'-monophosphate dehydrogenase 2) and two unidentified m/z values (1305.840 and 1661.060) that were significantly higher in patients with shorter survival (0-3 years relative to 4-6 years and 7+ years). This is the first study using MALDI-MSI, combined with LC-MS/MS, on a large cohort of CRLM patients to identify the spatial proteome in this malignancy. Further, we identify several protein candidates that may be suitable for drug targeting or for future prognostic biomarker development.
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Neoplasias Colorrectales , Neoplasias Hepáticas , Humanos , Masculino , Femenino , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteómica/métodos , Cromatografía Liquida/métodos , Proteoma , Espectrometría de Masas en Tándem , PéptidosRESUMEN
RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 µm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.
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Péptidos , Espectrometría de Masas en Tándem , Animales , Ratones , Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Polisacáridos/análisis , Pulmón/química , LípidosRESUMEN
N-glycan alterations contribute to the progression of several joint diseases, including knee osteoarthritis (KOA). However, molecular changes in KOA subchondral trabecular bone, when exposed to different joint loading forces, are still unknown. The aim of this study was, therefore, to demonstrate the feasibility to differentiate N-glycan changes in subchondral trabecular bone from four different joint loading forces of the tibial plateau regions (i.e., Lateral Anterior (L-A), Lateral Posterior (L-P), Medial Anterior (M-A), and Medial Posterior (M-P)) in KOA patients (n = 10) using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at 20 µm spatial resolution. The degree of cartilage degeneration was evaluated histologically, and the subchondral bone tissue microarrays (TMAs) were subsequently manually constructed from formalin-fixed paraffin-embedded (FFPE) KOA osteochondral (i.e., cartilage-subchondral bone) tissues. Overall, the Osteoarthritis Research Society International (OARSI) histological grade was significantly higher and the size of chondrocytes in the superficial zone was much larger for both M-A and M-P compared to L-A and L-P of cartilage (p = 0.006, p = 0.030, p = 0.028, and p = 0.010; respectively). Among the 65 putative N-glycans observed by MALDI-MSI, 2 core fucosylated bi-antennary N-glycans, m/z 1809.64; (Hex)5(HexNAc)4(Fuc)1 and 2100.73; (NeuAc)1(Hex)5(HexNAc)4(Fuc)1, were significantly higher in intensity in M-A compared to L-A of the trabecular bone (p = 0.027, and p = 0.038, respectively). These N-glycans were then further structurally characterized by in situ MS/MS fragmentation post-MALDI-MSI. Our results demonstrate, for the first time, N-glycan alterations can occur at different joint loading forces in the KOA tibial plateau and the feasibility of subchondral bone TMA construction for N-glycan MALDI-MSI.
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Osteoartritis de la Rodilla , Humanos , Osteoartritis de la Rodilla/diagnóstico por imagen , Osteoartritis de la Rodilla/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem , Polisacáridos/química , Tibia/patologíaRESUMEN
Abnormal N-glycosylation has been shown to play an important role in the pathogenesis of multiple diseases. However, little is known about the relationship between N-glycosylation and knee osteoarthritis (KOA) progression at the tissue level. Thus, the aim of this study was to quantify the cartilage histomorphometric changes in formalin-fixed paraffin-embedded (FFPE) tissue collected from the lateral and medial compartments of the tibial plateau KOA patients (n = 8). Subsequently, N-glycans were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) followed by in situ MS/MS fragmentation. Overall, the Osteoarthritis Research Society International (OARSI) histological grade and cartilage surface fibrillation index were significantly higher, and chondrocyte size in the superficial zone was much larger, for the medial high-loaded cartilage compared to the lateral less-loaded cartilage. Among 92 putative N-glycans observed by MALDI-MSI, 3 complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, and 1 oligomannose-type N-glycan, (Hex)9(HexNAc)2, were significantly higher in intensity in the medial cartilage compared to the lateral cartilage, whereas 2 tetra-antennary fucosylated-type N-glycans, (Hex)3(HexNAc)6(Fuc)2 and (Hex)3(HexNAc)6(Fuc)3, were significantly higher in intensity in the lateral cartilage than the medial cartilage. Our findings indicate that complex-type N-glycans are associated with higher severity of cartilage degeneration and may influence the cellular processes of KOA.
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Osteoartritis de la Rodilla , Humanos , Osteoartritis de la Rodilla/patología , Espectrometría de Masas en Tándem , Cartílago/química , Cartílago/patología , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Tetrodotoxin (TTX) is a potent marine neurotoxin that occurs in several Australian phyla, including pufferfish, toadfish, gobies, and the blue-ringed octopus. These animals are partially immune, and TTX is known to bioaccumulate and subject to trophic transfer. As such, it could be more ubiquitously distributed in animals than is currently known. Flatworms of the order Polycladida are commonly occurring invertebrates in intertidal ecosystems and are especially diverse in Australian waters. While TTX has been identified in polyclads from Japan and New Zealand, Australian species have yet to be tested. In this study, several eastern Australian polyclad flatworm species from the suborders Cotylea and Acotylea were tested for TTX and analogs by HILIC-HRMS to understand the distribution of this toxin within these suborders. Herein, we report the detection of TTX and some known analogs in polyclad species, one of which is a pest to shellfish aquaculture. We also report, for the first time, the application of MALDI mass spectrometry imaging utilized to map TTX spatially within the intestinal system of polyclads. The identification of TTX and its analogs in Australian flatworms illustrates a broader range of toxic flatworms and highlights that analogs are important to consider when studying the distributions of toxins in animals.
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Ecosistema , Platelmintos , Animales , Tetrodotoxina/química , Australia , Platelmintos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.
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Osteoartritis de la Rodilla , Humanos , Persona de Mediana Edad , Cromatografía Liquida , Espectrometría de Masas en Tándem , Polisacáridos/análisis , Cartílago/química , Formaldehído/química , BiomarcadoresRESUMEN
Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans.
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Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been successfully used to elucidate the relative abundance and spatial mapping of analytes in situ. Currently, sample preparation workflows for soft formalin-fixed paraffin-embedded (FFPE) tissues, such as brain, liver, kidney, and heart, have been successfully developed. However, hard tissues, such as cartilage-bone, tooth, and whole mouse body, have resulted in the loss of morphology or tissue during the heat-induced epitope retrieval (HIER) step on commercially available conductive indium tin oxide (ITO) slides. Therefore, we have successfully developed a novel and cost-effective sample preparation workflow in which commercial conductive ITO slides are pre-coated with gelatin and chromium potassium sulfate dodecahydrate to improve the adherence of FFPE human osteoarthritic cartilage-bone tissue sections. Gelatin-coated ITO slides also resulted in overall higher N-glycan signal intensity for not only FFPE osteoarthritic cartilage-bone tissue but also for FFPE hard-boiled egg white used as a quality control to assess the quality of sample preparation and MALDI-MSI acquisition. In summary, we present a novel straightforward workflow to improve slide adherence and morphological preservation of FFPE cartilage-bone tissue sections during HIER while improving the signal intensity of N-glycans spatially mapped from the same tissue sections by MALDI-MSI.
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Huesos/química , Cartílago/química , Osteoartritis/patología , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Anciano de 80 o más Años , Huesos/patología , Cartílago/patología , Femenino , Gelatina/química , Humanos , Compuestos de Estaño/químicaRESUMEN
Osteoarthritis (OA) is the most common degenerative joint disease, predicted to increase in incidence year by year due to an ageing population. Due to the biological complexity of the disease, OA remains highly heterogeneous. Although much work has been undertaken in the past few years, underlying molecular mechanisms leading to joint tissue structural deterioration are not fully understood, with only few validated markers for disease diagnosis and progression being available. Discovery and quantitation of various OA-specific biomarkers is still largely focused on the bodily fluids which does not appear to be reliable and sensitive enough. However, with the advancement of spatial proteomic techniques, several novel peptides and proteins, as well as N-glycans, can be identified and localised in a reliable and sensitive manner. To summarise the important findings from OA biomarker studies, papers published between 2000 and 2020 were searched via Google Scholar and PubMed. Medical subject heading (MeSH) terms 'osteoarthritis', 'biomarker', 'synovial fluid', 'serum', 'urine', 'matrix-assisted laser desorption/ionisation', 'mass spectrometry imaging', 'proteomic', 'glycomic', 'cartilage', 'synovium' AND 'subchondral bone' were selectively used. The literature search was restricted to full-text original research articles and written only in English. Two main areas were reviewed for OA biomarker studies: (1) an overview of disease-specific markers detected from different types of OA bio-samples, and (2) an up-to-date summary of the tissue-specific OA studies that have utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). Overall, these OA biomarkers could provide clinicians with information for better the diagnosis, and prognosis of individual patients, and ultimately help facilitate the development of disease-modifying treatments.
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Biomarcadores/metabolismo , Cartílago Articular/patología , Osteoartritis/patología , Proteoma/análisis , Proteoma/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Membrana Sinovial/patología , Cartílago Articular/metabolismo , Humanos , Osteoartritis/metabolismo , Membrana Sinovial/metabolismoRESUMEN
The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during sample preparation can be used to improve the mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for the quality check of sample preparation and data acquisition, which is particularly relevant when either many spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).
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Clara de Huevo/química , Neoplasias Endometriales/química , Fragmentos de Péptidos/análisis , Polisacáridos/análisis , Proteínas/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normas , Secuencia de Aminoácidos , Neoplasias Endometriales/diagnóstico , Neoplasias Endometriales/patología , Femenino , Humanos , Microtomía , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Proteínas/química , Proteolisis , Control de Calidad , Análisis de Matrices Tisulares , Adhesión del Tejido , Tripsina/químicaRESUMEN
The application of proteomic liquid chromatography mass spectrometry (LC-MS) for identifying proteins and peptides associated with human disease is rapidly growing in clinical diagnostics. However, the ability to accurately and consistently detect disease-associated peptides remains clinically uncertain. Variability in diagnostic testing occurs in part due to the absence of appropriate reference testing materials and standardised clinical guidelines for proteomic testing. In addition, multiple proteomic testing pipelines have not been fully assessed through external quality assurance (EQA). This trial was therefore devised to evaluate the performance of a small number of mass spectrometry (MS) testing facilities to (i) evaluate the EQA material for potential usage in a proteomic quality assurance program, and to (ii) identify key problem areas associated with human peptide testing. Five laboratories were sent six peptide reference testing samples formulated to contain a total of 35 peptides in differing ratios of light (natural) to heavy (labelled) peptides. Proficiency assessment of laboratory data used a modified approach to similarity and dissimilarity testing that was based on Bray-Curtis and Sorensen indices. Proficiency EQA concordant consensus values could not be derived from the assessed data since none of the laboratories correctly identified all reference testing peptides in all samples. However, the produced data may be reflective of specific inter-laboratory differences for detecting multiple peptides since no two testing pipelines used were the same for any laboratory. In addition, laboratory feedback indicated that peptide filtering of the reference material was a common key problem area prior to analysis. These data highlight the importance of an EQA programme for identifying underlying testing issues so that improvements can be made and confidence for clinical diagnostic analysis can be attained.
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Péptidos/análisis , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Humanos , Proteómica/métodos , Proteómica/normas , Control de Calidad , Estándares de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normasRESUMEN
While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.
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Manosa/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Progresión de la Enfermedad , Glicosilación , Humanos , Espectrometría de Masas en TándemRESUMEN
Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)6 + (Man)3 (GlcNAc)2 , and the complex neutral structure, (Hex)2 (HexNAc)2 (Deoxyhexose)1 + (Man)3 (GlcNAc)2 . The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients.
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Biomarcadores de Tumor/genética , Cistadenoma Seroso/genética , Neoplasias Ováricas/genética , Polisacáridos/genética , Biomarcadores de Tumor/química , Cromatografía Liquida , Cistadenoma Seroso/patología , Femenino , Genómica/métodos , Glicosilación , Humanos , Imagen Molecular , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Microambiente Tumoral/genéticaRESUMEN
Protein glycosylation, particularly N-linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell-cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS-based techniques, to qualitatively and quantitatively assess N-glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC-ESI-MS/MS and MALDI time-of-flight MS (MALDI-TOF-MS), which have been used to analyze clinical samples, such as serum, plasma, ascites, and tissue. Targeting the aberrant N-glycosylation patterns observed in MALDI-MS imaging (MSI) offers a platform to visualize N-glycans in tissue-specific regions. The studies on the intra-patient (i.e., a comparison of tissue-specific regions from the same patient) and inter-patient (i.e., a comparison of tissue-specific regions between different patients) variation of early- and late-stage ovarian cancer (OC) patients identify specific N-glycan differences that improve understanding of the tumor microenvironment and potentially improve therapeutic strategies for the clinic.
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Técnicas de Química Analítica , Neoplasias Ováricas/metabolismo , Polisacáridos/metabolismo , Femenino , Humanos , Espectrometría de MasasRESUMEN
Provided is the annotated raw data for N-glycan mass spectrometry imaging (MSI) annotations in thin cross-sections of formalin-fixed and paraffin-embedded murine kidney. Relevant meta-data have been provided in this brief and the raw MSI data can be accessed using ProteomeXchange with the PRoteomics IDEntifications (PRIDE) identifier PXD009808. This brief is the first in a set of submissions from our group which will make raw data publicly accessible for existing and future MSI studies.
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RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of the proteome of a tissue has been an established technique for the past decade. In the last few years, MALDI-MSI of the N-glycome has emerged as a novel MALDI-MSI technique. To assess the accuracy and clinical significance of the N-linked glycan spatial distribution, we have developed a method that utilises MALDI-MSI followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) in order to assign glycan structures to the differentiating MALDI-MSI glycan masses released from the tissue glycoproteins. METHODS AND RESULTS: Our workflow presents a comprehensive list of instructions on how to (i) apply MALDI-MSI to spatially map the N-glycome across formalin-fixed paraffin-embedded (FFPE) clinical samples, (ii) structurally characterise N-glycans extracted from consecutive FFPE tissue sections by LC/MS/MS, and (iii) match relevant N-glycan masses from MALDI-MSI with confirmed N-glycan structures determined by LC/MS/MS. CONCLUSIONS: Our protocol provides groups that are new to this technique with instructions how to establish N-glycan MALDI-MSI in their laboratory. Furthermore, the method assigns N-glycan structural detail to the masses obtained in the MALDI-MS image. Copyright © 2017 John Wiley & Sons, Ltd.
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Ovarian cancer is a fatal gynaecological malignancy in adult women with a five-year overall survival rate of only 30%. Glycomic and glycoproteomic profiling studies have reported extensive protein glycosylation pattern alterations in ovarian cancer. Therefore, spatio-temporal investigation of these glycosylation changes may unearth tissue-specific changes that occur in the development and progression of ovarian cancer. A novel method for investigating tissue-specific N-linked glycans is using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) on formalin-fixed paraffin-embedded (FFPE) tissue sections that can spatially profile N-glycan compositions released from proteins in tissue-specific regions. In this study, tissue regions of interest (e.g. tumor, stroma, adipose tissue and necrotic areas) were isolated from FFPE tissue sections of advanced serous ovarian cancers (n = 3). PGC-LC-ESI-MS/MS and MALDI-MSI were used as complementary techniques to firstly generate structural information on the tissue-specific glycans in order to then obtain high resolution images of the glycan structure distribution in ovarian cancer tissue. The N-linked glycan repertoires carried by the proteins in these tissue regions were structurally characterized for the first time in FFPE ovarian cancer tissue regions, using enzymatic peptide-N-glycosidase F (PNGase F) release of N-glycans. The released glycans were analyzed by porous graphitized carbon liquid chromatography (PGC-LC) and collision induced electrospray negative mode MS fragmentation analysis. The N-glycan profiles identified by this analysis were then used to determine the location and distribution of each N-glycan on FFPE ovarian cancer sections that were treated with PNGase F using high resolution MALDI-MSI. A tissue-specific distribution of N-glycan structures identified particular regions of the ovarian cancer sections. For example, high mannose glycans were predominantly expressed in the tumor tissue region whereas complex/hybrid N-glycans were significantly abundant in the intervening stroma. Therefore, tumor and non-tumor tissue regions were clearly demarcated solely on their N-glycan structure distributions.
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Neoplasias Ováricas/metabolismo , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Femenino , Glicómica/métodos , Humanos , Especificidad de Órganos , Adhesión en Parafina , Polisacáridos/química , Proteómica/métodos , Fijación del TejidoRESUMEN
Magnetic resonance imaging (MRI) is a non-invasive technique routinely used to investigate pathological changes in knee osteoarthritis (OA) patients. MRI uniquely reveals zones of the most severe change in the subchondral bone (SCB) in OA, called bone marrow lesions (BMLs). BMLs have diagnostic and prognostic significance in OA, but MRI does not provide a molecular understanding of BMLs. Multiple N-glycan structures have been observed to play a pivotal role in the OA disease process. We applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of N-glycans to formalin-fixed paraffin-embedded (FFPE) SCB tissue sections from patients with knee OA, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was conducted on consecutive sections to structurally characterize and correlate with the N-glycans seen by MALDI-MSI. The application of this novel MALDI-MSI protocol has enabled the first steps to spatially investigate the N-glycome in the SCB of knee OA patients.
Asunto(s)
Cartílago/diagnóstico por imagen , Osteoartritis de la Rodilla/diagnóstico por imagen , Polisacáridos/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Anciano , Médula Ósea/patología , Cartílago/química , Cartílago/patología , Cromatografía Liquida/métodos , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Imagen Molecular/métodos , Osteoartritis de la Rodilla/diagnóstico , Osteoartritis de la Rodilla/patología , Polisacáridos/química , Tibia/diagnóstico por imagen , Tibia/patologíaRESUMEN
Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies.