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Fulltext N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues

Everest-Dass AV, Briggs MT, Kaur G, Oehler MK, Hoffmann P, Packer NH

Mol Cell Proteomics, 2016 09;15(9):3003-16.
PMID: 27412689 DOI: 10.1074/mcp.M116.059816

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.
Altered N-linked glycosylation in endometrial cancer

Mittal P, Briggs M, Klingler-Hoffmann M, Kaur G, Packer NH, Oehler MK, et al.

Anal Bioanal Chem, 2021 Apr;413(10):2721-2733.
PMID: 33222001 DOI: 10.1007/s00216-020-03039-z

It is well established that cell surface glycans play a vital role in biological processes and their altered form can lead to carcinogenesis. Mass spectrometry-based techniques have become prominent for analysing N-linked glycans, for example using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Additionally, MALDI MS can be used to spatially map N-linked glycans directly from cancer tissue using a technique termed MALDI MS imaging (MALDI MSI). This powerful technique combines mass spectrometry and histology to visualise the spatial distribution of N-linked glycans on a single tissue section. Here, we performed N-glycan MALDI MSI on six endometrial cancer (EC) formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) consisting of eight EC patients with lymph node metastasis (LNM) and twenty without LNM. By doing so, several putative N-linked glycan compositions were detected that could significantly distinguish normal from cancerous endometrium. Furthermore, a complex core-fucosylated N-linked glycan was detected that could discriminate a primary tumour with and without LNM. Structural identification of these putative N-linked glycans was performed using porous graphitized carbon liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS). Overall, we observed higher abundance of oligomannose glycans in tumour compared to normal regions with AUC ranging from 0.85-0.99, and lower abundance of complex N-linked glycans with AUC ranges from 0.03-0.28. A comparison of N-linked glycans between primary tumours with and without LNM indicated a reduced abundance of a complex core-fucosylated N-glycan (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2, in primary tumour with associated lymph node metastasis. In summary, N-linked glycan MALDI MSI can be used to differentiate cancerous endometrium from normal, and endometrial cancer with LNM from endometrial cancer without.
N-Glycan matrix-assisted laser desorption/ionization mass spectrometry imaging protocol for formalin-fixed paraffin-embedded tissues

Briggs MT, Ho YY, Kaur G, Oehler MK, Everest-Dass AV, Packer NH, et al.

Rapid Commun Mass Spectrom, 2017 May 30;31(10):825-841.
PMID: 28271569 DOI: 10.1002/rcm.7845

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.
MALDI Mass Spectrometry Imaging of Early- and Late-Stage Serous Ovarian Cancer Tissue Reveals Stage-Specific N-Glycans

Briggs MT, Condina MR, Ho YY, Everest-Dass AV, Mittal P, Kaur G, et al.

Proteomics, 2019 11;19(21-22):e1800482.
PMID: 31364262 DOI: 10.1002/pmic.201800482

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.
Translating N-Glycan Analytical Applications into Clinical Strategies for Ovarian Cancer

Briggs MT, Condina MR, Klingler-Hoffmann M, Arentz G, Everest-Dass AV, Kaur G, et al.

Proteomics Clin Appl, 2019 05;13(3):e1800099.
PMID: 30367710 DOI: 10.1002/prca.201800099

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.