Efficient Procedure for N-Glycan Analyses and Detection of Endo H-Like Activity in Human Tumor Specimens

Abstract

Although the importance of glycosylation has been thoroughly recognized in association with a number of biological processes, efficient assessments of glycans have been hampered by both the limited size of specimens and lengthy sample preparations, particularly in clinical settings. Here we report a simple preparative method for N-glycan analyses. It involves only short one-step chloroform-methanol extraction in presence or absence of water prior to PNGase F deglycosylation. The procedure was successfully applied to the investigation of N-glycans obtained from small numbers of in vitro cultured cancer cells (≤1 × 10(5)) and to tumor tissues, including patient biopsies of small size. MALDI-MS analysis confirmed the efficient release of all N-glycan types including complex forms with poly-N-acetyllactosamine chains. In addition, nonaqueous extraction of specimens from several established cancer cell lines, as well as patient tumor tissues, yielded high-mannose glycans with one GlcNAc moiety (Man3-9GlcNAc), strongly suggesting preservation of enzymatic activity analogous to Endo H enzyme. In summary, the method is both a step toward the practical use of glycan profiling and a way to detect Endo H-like activity in cancer specimens.

N-Glycan profiling of lung adenocarcinoma in patients at different stages of disease

“…We reported earlier sample preparative procedure for investigation of N-glycans from human cells of established cancer cell lines including those derived from LAC preserved by lyophilization [31]. The aim of the present study was to investigate applicability of this method in clinical settings for analysis of N-glycans from fresh as well as defrosted tissues of LAC patients, and to determine potential correlations between N-glycan profiles and disease stages of LAC.…”

“…Prior to deglycosylation, washed tissues were first extracted with organic solvent as described previously [31]. Briefly, 1-2 tissue pieces with size 1-3 mm were mixed with 300 µL solution consisting of chloroform-methanol-water (8:4:1) and sonicated for 3 min, then shortly centrifuged and supernatant was discarded.…”

“…SPE purified glycans in deionized water (10-80 μL) underwent on-target derivatization with PHN as described previously [31,33]. Briefly, matrix (0.8 μL) consisting of 2aza-2-thiothymine and phenylhydrazine hydrochloride (ATT/PHN.HCl, 2:1) with the final concentration 3% in ACN/deionized water (1:1) was spotted on the surface of the AnchorChip target and followed by addition of purified glycan fraction (1.5 μL).…”

“…The main purpose of our present work was to investigate the validity of N-glycan changes in tumor and noncancerous tissues and discriminate differences related to disease stages of LAC. Previously, we reported a simple preparative procedure, which enabled effective release of all N-glycan types from freeze-dried small-sized specimens including two patient biopsies [31]. Using this approach on nonprocessed samples, we determined N-glycan profiles of fresh tumor versus adjacent noncancerous tissues from patients with LAC at the disease stage I-III, and evaluated potential applicability of the data from this study to small size biopsies procured by bronchoscopy, which usually lack control (noncancerous) tissue samples.…”

A facile method for cellular N-glycomic profiling by matrix-assisted laser desorption/ionization mass spectrometry

“…However, use of CMW showed higher signal intensities, which might result from the better protein recovery. 19 In addition, less impurity peaks were observed in the low m/z range of mass spectrum by using CMW. As a result, the choice of extraction method was CMW for further experiments.…”