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 paraffinembedded ( Ovarian cancer is the fifth most fatal malignancy in adult women with an estimated 21,290 new cases diagnosed and 14,180 deaths recorded in the United States during 2015 (1). There are several reasons for the poor prognosis of ovarian cancer and its diagnosis at advanced stage-lack of diagnostic markers for the early detection (2, 3), rapid metastasis of the disease (4), and limited or modest understanding of the etiology, origin and the diverse clinical and pathological behavior of the tumors (5). Moreover, epithelial ovarian cancer comprises of several distinct sub-types based on their histopathological features into serous, endometrioid, clear-cell, mucinous, and undifferentiated subtypes (6, 7).Protein glycosylation is an important post-translational modification which has relevance in many biological processes such as cell signaling, immune responses, extracellular interaction and cell adhesion (8, 9). Aberrant protein glycosylation such as the expression of truncated glycans as well From the ‡Faculty

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.Graphical AbstractMALDI imaging MS of N-linked glycans released from formalin-fixed paraffin-embedded murine kidney sections. Ion intensity maps for (Hex)2(HexNAc)3(Deoxyhexose)3+(Man)3(GlcNAc)2 (m/z 2304.932, red), (Hex)6+(Man)3(GlcNAc)2 (m/z 1905.742, green) and (Hex)2(HexNAc)2+(Man)3(GlcNAc)2 (m/z 1663.756, blue)Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-8293-7) contains supplementary material, which is available to authorized users.

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 [Man1‐3GlcNAc2Fuc0‐1], 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 Man2‐3GlcNAc2Fuc1, 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‐β‐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.

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.