Dataset Information

Glycomic profiling of tissue sections by LC-MS.

ABSTRACT: Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to online purification and LC-electrospray ionization (ESI)-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-?L aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak areas of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed.

SUBMITTER: Hu Y

PROVIDER: S-EPMC3673026 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Glycomic profiling of tissue sections by LC-MS.

Hu Yunli Y Zhou Shiyue S Khalil Sarah I SI Renteria Calvin L CL Mechref Yehia Y

Analytical chemistry 20130326 8

Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples fi ...[more]

PMID: 23438902

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Project description:Determination of disease-relevant proteomic profiles from limited tissue specimens, such as pathological biopsies and tissues from small model organisms, remains an analytical challenge and a much needed clinical goal. In this study, a transgenic mouse disease model of cardiac-specific H-Ras-G12V induced hypertrophic cardiomyopathy provided a system to explore the potential of using mass spectrometry (MS)-based proteomics to obtain a disease-relevant molecular profile from amount-limited specimens that are routinely used in pathological diagnosis. Our method employs a two-stage methanol-assisted solubilization to digest lysates prepared from 8-μm-thick fresh-frozen histological tissue sections of diseased/experimental and normal/control hearts. Coupling this approach with a nanoflow reversed-phase liquid chromatography (LC) and a hybrid linear ion trap/Fourier transform-ion cyclotron resonance MS resulted in the identification of 704 and 752 proteins in hypertrophic and wild-type (control) myocardium, respectively. The disease driving H-Ras protein along with vimentin were unambiguously identified by LC-MS in hypertrophic myocardium and cross-validated by immunohistochemistry and western blotting. The pathway analysis involving proteins identified by MS showed strong association of proteomic data with cardiovascular disease. More importantly, the MS identification and subsequent cross-validation of Wnt3a and β-catenin, in conjunction with IHC identification of phosphorylated GSK-3β and nuclear localization of β-catenin, provided evidence of Wnt/β-catenin canonical pathway activation secondary to Ras activation in the course of pathogenic myocardial hypertrophic transformation. Our method yields results indicating that the described proteomic approach permits molecular discovery and assessment of differentially expressed proteins regulating H-Ras induced hypertrophic cardiomyopathy. Selected proteins and pathways can be further investigated using immunohistochemical techniques applied to serial tissue sections of similar or different origin.

Dataset Information

Glycomic profiling of tissue sections by LC-MS.

Publications

Glycomic profiling of tissue sections by LC-MS.

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