title: Universal Solid-Phase Reversible Sample-Prep for Concurrent Proteome and N-Glycome Characterization.
abstract: We describe a novel solid-phase reversible sample-prep (SRS) platform that enables rapid sample preparation for concurrent proteome and N-glycome characterization for nearly all protein samples. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal to no affinity for peptides and other small molecules. By leveraging this inherent size difference between proteins and peptides, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, peptides, etc.), extensive manipulation including enzymatic and chemical treatments on bead-bound proteins, and easy recovery of N-glycans and peptides. SRS was evaluated in a wide range of samples including glycoproteins, cell lysate, murine tissues, and human urine. SRS was also coupled to a quantitative strategy to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Previous studies suggested that DIAPH3 silencing in DU145 induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this pilot study we identified distinct proteomic and N-glycomic alterations between them. A metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly up-regulated in DIAPH3-silenced cells, indicating a possible connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting cross-links between DIAPH3 and glycosyltransferase networks.
authors.affiliation: Department of Urology, Boston Children's Hospital and Harvard Medical School , 300 Longwood Avenue, Boston, Massachusetts 02115, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Department of Urology, Boston Children's Hospital and Harvard Medical School , 300 Longwood Avenue, Boston, Massachusetts 02115, United States.
authors.forename: Samantha
authors.initials: S
authors.lastname: Morley
authors.affiliation: Department of Urology, Boston Children's Hospital and Harvard Medical School , 300 Longwood Avenue, Boston, Massachusetts 02115, United States.
authors.forename: Stephen
authors.initials: S
authors.lastname: Kostel
authors.affiliation: Division of Cancer Biology and Therapeutics, Cedars-Sinai Medical Center , Los Angeles, California 90048, United States.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Freeman
authors.affiliation: EMD Millipore Corporation , Billerica, Massachusetts 01821, United States.
authors.forename: Vivek
authors.initials: V
authors.lastname: Joshi
authors.affiliation: EMD Millipore Corporation , Billerica, Massachusetts 01821, United States.
authors.forename: David
authors.initials: D
authors.lastname: Brewster
authors.affiliation: Department of Urology, Boston Children's Hospital and Harvard Medical School , 300 Longwood Avenue, Boston, Massachusetts 02115, United States.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Lee
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D000080865:Formins; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008810:Mice, Inbred C57BL; D011485:Protein Binding; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D036104:Receptor, EphA2 / Q000235:genetics / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C484571:DIAPH3 protein, human; D000080865:Formins; D006023:Glycoproteins; D020543:Proteome; D012822:Silicon Dioxide; D036104:Receptor, EphA2
keywords: N-glycome; glycoprotein; membrane protein; plasma; proteome; reversible solid phase; sample-prep; urine