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1.
J Proteome Res ; 13(2): 961-8, 2014 Feb 07.
Article in English | MEDLINE | ID: mdl-24303873

ABSTRACT

In clinical settings, biopsies are routinely used to determine cancer type and grade based on tumor cell morphology, as determined via histochemical or immunohistochemical staining. Unfortunately, in a significant number of cases, traditional biopsy results are either inconclusive or do not provide full subtype differentiation, possibly leading to inefficient or ineffective treatment. Glycomic profiling of the cell membrane offers an alternate route toward cancer diagnosis. In this study, isomer-sensitive nano-LC/MS was used to directly obtain detailed profiles of the different N-glycan structures present on cancer cell membranes. Membrane N-glycans were extracted from cells representing various subtypes of breast, lung, cervical, ovarian, and lymphatic cancer. Chip-based porous graphitized carbon nano-LC/MS was used to separate, identify, and quantify the native N-glycans. Structure-sensitive N-glycan profiling identified hundreds of glycan peaks per cell line, including multiple isomers for most compositions. Hierarchical clusterings based on Pearson correlation coefficients were used to quickly compare and separate each cell line according to originating organ and disease subtype. Based simply on the relative abundances of broad glycan classes (e.g., high mannose, complex/hybrid fucosylated, complex/hybrid sialylated, etc.), most cell lines were readily differentiated. More closely related cell lines were differentiated based on several-fold differences in the abundances of individual glycans. Based on characteristic N-glycan profiles, primary cancer origins and molecular subtypes could be distinguished. These results demonstrate that stark differences in cancer cell membrane glycosylation can be exploited to create an MS-based biopsy, with potential applications toward cancer diagnosis and direction of treatment.


Subject(s)
Neoplasms/pathology , Polysaccharides/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Chromatography, Liquid , Glycomics , Humans , Mass Spectrometry , Neoplasms/classification , Neoplasms/metabolism
2.
Bioanalysis ; 5(5): 545-59, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23425271

ABSTRACT

BACKGROUND: Erythropoietin is a therapeutic glycoprotein that stimulates red blood cell production. The quality, safety and potency of recombinant erythropoietins are determined largely by their glycosylation. Small variations in cell culture conditions can significantly affect the glycosylation, and therefore the efficacy, of recombinant erythropoietins. Thus, detailed glycomic analyses are necessary to assess biotherapeutic quality. We have developed a platform for qualitative and quantitative glycomic analysis of recombinant erythropoietins. RESULTS: The platform was used to profile native N-glycans from three production batches of darbepoetin alfa (also known as NESP), a common form of recombinant erythropoietin. Darbepoetin alfa was found to contain an abundance of large, multi-antennary N-glycans with high levels of sialylation, O-acetylation and dehydration. Results were verified by independent orthogonal analysis with both MALDI-TOF and nano-LC/Q-TOF MS. CONCLUSION: This platform may be applied to QC and batch analysis of not only recombinant erythropoietin, but also other complex, glycosylated biotherapeutics and biosimilars.


Subject(s)
Biosimilar Pharmaceuticals/analysis , Erythropoietin/analysis , Polysaccharides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Biosimilar Pharmaceuticals/standards , Carbohydrate Conformation , Erythropoietin/genetics , Erythropoietin/standards , Glycosylation , Humans , Isomerism , Nanotechnology , Neuraminic Acids/analysis , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Polysaccharides/isolation & purification , Polysaccharides/standards , Quality Control , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/standards , Solid Phase Extraction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/standards
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