Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. Breast cancer is the leading cause of cancer death and the most frequently diagnosed cancer among women worldwide (1). In the United States alone, ϳ40,000 deaths and 210,000 new cases were expected in 2010 (2). Incidence rates continue to rise especially in many developing and westernized countries. Unfortunately, early stages of breast cancer show no noticeable symptoms. Early diagnosis is critical because the chance of survival is greater in early stage (Stage I and II) breast cancer. It is estimated that 98% of U. S. women will survive longer than 5 years if the cancer is detected early. At late stages (Stages III and IV), however, only 28% will survive longer than 5 years (1).Currently, carbohydrate antigen 15-3 (CA 15-3) is the most common clinical serum marker for breast cancer. This marker uses immunoassay to detect MUC-1, a mucin glycoprotein overexpressed with breast cancer. Other markers for breast cancer include carcinoembryonic antigen, an anchored glycoprotein involved in cell adhesion, and CA 27.29, another MUC-1-derived glycoprotein marker. A common feature of these three markers is that all are proteins containing glycoforms.However, the current markers described above are not recommended by the American Society of Clinical Oncology as markers for screening, diagnostic, or staging tests for breast cancer (3). During the early stages of breast cancer, the sensitivity (i.e. patients correctly identified) of these markers is less than 25% (3-7). Moreover, the specificity (i.e. people without cancer correctly identified) is also problematic: up to 20%-30% of women without breast cancer, i.e. healthy individuals, women with benign breast lesions, people with benign diseases such as liver disease, and people with other types of advanced adenocarcinoma, have elevated levels of the said markers (3-7). Thus, an elevated marker level is not specific to breast cancer and may lead to false positive diagnoses for the healthy individual.Aberrant glycosylation is observed in the progression of many types of diseases, including different cancers (8, 9). Glycosylation, one of the most common forms of post-translational modification, is highly sensitive to ...

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Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution mass spectrometry, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan based markers.

SummaryHuman milk contains approximately 200 complex oligosaccharides believed to stimulate the growth and establishment of a protective microbiota in the infant gut. The lack of scalable analytical techniques has hindered the measurement of bacterial metabolism of these and other complex prebiotic oligosaccharides. An in vitro, multi‐strain, assay capable of measuring kinetics of bacterial growth and detailed oligosaccharide consumption analysis by FTICR‐MS was developed and tested simultaneously on 12 bifidobacterial strains. For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used. A custom software suite developed in house called Glycolyzer was used to process the large amounts of oligosaccharide mass spectra automatically with 13C corrections based on de‐isotoping protocols. High growth on HMOs was characteristic of Bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains tested, B. longum bv. longum, B. adolescentis, B. breve and B. bifidum, showed low or only moderate growth ability. Total oligosaccharide consumption ranged from a high of 87% for B. infantis JCM 7009 to only 12% for B. adolescentis ATCC 15703. A detailed analysis of consumption glycoprofiles indicated strain‐specific capabilities towards differential metabolism of milk oligosaccharides. This method overcomes previous limitations in the quantitative, multi‐strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides.

Glycans constitute a new class of compounds for biomarker discovery. Glycosylation is a common post-translational modification and is often associated with transformation to malignancy. To analyze glycans, they are released from proteins, enriched, and measured with mass spectrometry. For biomarker discovery, repeatability at every step of the process is important. Locating and minimizing the process variability is key to establishing a robust platform stable enough for biomarker discovery. Understanding the variability of the measurement devices helps understand the variability associated with the chemical processing. This report explores the potential use of methods expediting the enzymatic release of glycans such as a microwave reactor and automation of the solid-phase extraction with a robotic liquid handler. The study employs matrix-assisted laser desorption/ ionization -Fourier transform ion cyclotron resonance mass spectrometry but would be suitable with any mass spectrometry method. Methods for system-wide data analysis are examined because proper metrics for evaluating the performance of glycan sample preparation procedures are not well established.