Glycomic Profiling of Tissue Sections by LC-MS

Hu, Yunli; Zhou, Shiyue; Khalil, Sarah I.; Renteria, Calvin L.; Mechref, Yehia

doi:10.1021/ac400106x

Cited by 33 publications

(45 citation statements)

References 43 publications

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“…Dynamic exclusion was enabled to exclude ions repeated within 20 seconds. The energy used for HCD was 45% normalized collision energy optimized in previous studies (75–77) while 30% normalized collision energy was used for CID which was also previously optimized (78). A mass tolerance of 10 ppm was used for extracted ion chromatograms (EICs) of full MS data.…”

Section: Methodsmentioning

confidence: 99%

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

et al. 2016

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The biosynthesis of glycans is a template free process, hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS) based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated, thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages.

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Section: Methodsmentioning

confidence: 99%

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

et al. 2016

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“…Analysis of glycans from tissue surfaces has been previously demonstrated for N -glycans, 20,21 O -glycans, 21 and glycosaminoglycans (GAGs). 22–25,19 Similar to proteins, N -glycans can be analyzed by LC-MS following on-surface enzymatic digestion and extraction.…”

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confidence: 99%

Workflow for Combined Proteomics and Glycomics Profiling from Histological Tissues

et al. 2014

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Extracellular matrixes comprise glycoproteins, glycosaminoglycans and proteoglycans that order the environment through which cells receive signals and communicate. Proteomic and glycomic molecular signatures from tissue surfaces can add diagnostic power to the immunohistochemistry workflows. Acquired in a spatially resolved manner, such proteomic and glycomic information can help characterize disease processes and be easily applied in a clinical setting. Our aim toward obtaining integrated omics datasets was to develop the first workflow applicable for simultaneous analysis of glycosaminoglycans, N-glycans and proteins/peptides from tissue surface areas as small as 1.5 mm in diameter. Targeting small areas is especially important in the case of glycans, as their distribution can be very heterogeneous between different tissue regions. We first established reliable and reproducible digestion protocols for the individual compound classes by applying standards on the tissue using microwave irradiation to achieve reduced digestion times. Next, we developed a multienzyme workflow suitable for analysis of the different compound classes. Applicability of the workflow was demonstrated on serial mouse brain and liver sections, both fresh frozen and formalin-fixed. The glycomics data from the 1.5 mm diameter tissue surface area was consistent with data published on bulk mouse liver and brain tissues, which demonstrates the power of the workflow in obtaining combined molecular signatures from very small tissue regions.

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“…As the injection amount of reduced and permethylated RNase B reached as low as one ng, all five peaks associated with RNase B were detected. The sensitivity of RNase B is higher than our previous reported work of permethylated N-glycan analyzed by the data dependant mode of the Orbitrap mass spectometer [61]. The high sensitivity of this method might be attributed to the two stage mass filter of MRM, which decrease the chemical noise [62].…”

Section: Resultsmentioning

confidence: 91%

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

Zhou

Desantos-Garcia

et al. 2015

J. Am. Soc. Mass Spectrom.

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The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan structures was determined to be 30% while it was found to be 35% for either fucosylated or sialylated structures The optimum CE for mannose and complex type N-glycan structures was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan structures in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these structures was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitudes. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan structures enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

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Glycomic Profiling of Tissue Sections by LC-MS

Cited by 33 publications

References 43 publications

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

Workflow for Combined Proteomics and Glycomics Profiling from Histological Tissues

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

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