title: Development of an online microbore hollow fiber enzyme reactor coupled with nanoflow liquid chromatography-tandem mass spectrometry for global proteomics.
abstract: In this study, we report the development of a microbore hollow fiber enzyme reactor (mHFER) coupled to nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) for the online digestion or selective enrichment of glycopeptides and analysis of proteins. With mHFER, enzymatic digestion of protein could be achieved by continuous flow within a very small volume (~10 μL) of mHF inserted in a PEEK tube. Digested peptides exited through the pores of the hollow fiber membrane wall to external single or multiplexed trap columns for nLC-ESI-MS/MS analysis. Evaluation of online mHFER-nLC-ESI-MS/MS system was made with bovine serum albumin (BSA) by varying the temperature of digestion and the amount of protein injected. We evaluated the ability of the mHFER system to enrich glycopeptides by injecting a mixture of lectin (concanavalin A) and digested peptides from α-1-acid glycoprotein (AGP) into the mHFER, followed by delivery of PNGase F for endoglycosidic digestion. Nonglycosylated peptides unbound to lectins eluted at the first breakthrough run while N-linked glycopeptides eluted after the endoglycosidic digestion. The developed method was applied to urine samples from patients with prostate cancer and controls; 67 N-linked glycopeptides were identified and relative differences in glycopeptide content between patient and control samples were determined.
authors: Department of Chemistry, Yonsei University, Seoul, 120-749, Korea.
authors: Jin Yong
authors: JY
authors: Kim
authors: Sun Young
authors: SY
authors: Lee
authors: Sook-Kyung
authors: SK
authors: Kim
authors: Sang Ryoul
authors: SR
authors: Park
authors: Dukjin
authors: D
authors: Kang
authors: Myeong Hee
authors: MH
authors: Moon
mesh_terms: D054392:Adipokines; D019149:Bioreactors; D002352:Carrier Proteins / Q000652:urine; D016022:Case-Control Studies; D002853:Chromatography, Liquid / Q000379:methods*; D003208:Concanavalin A / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis* / Q000652:urine; D006801:Humans; D008297:Male; D009961:Orosomucoid / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011467:Prostate / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000652:urine; D040901:Proteomics; D012710:Serum Albumin, Bovine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000090203:Zn-Alpha-2-Glycoprotein
chemical_list: D054392:Adipokines; D002352:Carrier Proteins; D003208:Concanavalin A; D006020:Glycopeptides; D006023:Glycoproteins; D009961:Orosomucoid; D010446:Peptide Fragments; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012710:Serum Albumin, Bovine; C488891:AZGP1 protein, human; D000090203:Zn-Alpha-2-Glycoprotein