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Structural Identification of a Non-Glycosylated Variant at Ser126 for O-Glycosylation Site from EPO BRP, Human Recombinant Erythropoietin by LC/MS Analysis 원문보기

Molecules and cells, v.38 no.6, 2015년, pp.496 - 505  

Byeon, Jaehee (Deptatment of Stereoscopic Media, Korean German Institute of Technology) ,  Lim, Yu-Ri (BIOnSYSTEMS, Inc., R&D Center) ,  Kim, Hyong-Ha (Center for Bioanalysis, Korea Research Institute of Standards and Science) ,  Suh, Jung-Keun (Deptatment of Stereoscopic Media, Korean German Institute of Technology)

Abstract ▼ AI-Helper 아이콘AI-Helper

A variant peak was detected in the analysis of RP-HPLC of rHu-EPO, which has about 7% relative content. Fractions of the main and the variant peaks were pooled separately and further analyzed to identify the molecular structure of the variant peak. Total mass analysis for each peak fraction using ES...

주제어

#erythropoietin   #O-glycosylation   #total mass analysis   #Peptide Mapping  

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제안 방법

  • To clarify the difference of molecular masses between Peak M and Peak V, peptide mapping analysis was carried out using UPLC/ESI-MS. First, a method for peptide mapping for rHu- EPO was developed for the UPLC with on-line Q-TOF system to check if glycopeptides having O-glycosylation can be detected. Peptide mapping was carried out with endo-protease Asp-N or Glu-C treatment and subsequent separation of peptide fragments by RP-UPLC.
  • 6  250 mm). For quantification, RP-HPLC was performed using the following gradient conditions: solvent A was 0.1% TFA and solvent B was 0.1% TFA, 10% water, and 90% ACN. Protein samples were injected with the volume of 100 l and a flow rate of 0.
  • First, a method for peptide mapping for rHu- EPO was developed for the UPLC with on-line Q-TOF system to check if glycopeptides having O-glycosylation can be detected. Peptide mapping was carried out with endo-protease Asp-N or Glu-C treatment and subsequent separation of peptide fragments by RP-UPLC. The peptide fragments were identified by UV monitoring and on-line Q-TOF mass spectrometry.
  • RP-HPLC analysis was carried out using a Waters Alliance HPLC system equipped with a Waters 2489 UV/Visible detector (Waters, UK). Peak profiling was carried out using a VYDAC214TP54 column (C4, 300A, 5 m, 4.
  • To identify the structural characteristics of each peak, fractions having corresponding peaks were eluted and collected. The collected fractions were analyzed using ESI-MS to determine molecular weight for those peaks.
  • We confirmed that the method developed for peptide map-ping analysis of rHu-EPO is suitable for identifying D16 and E14 peptides with or without O-glycosylation. This method for peptide mapping was applied to the fractions having corresponding peaks from RP-HPLC analysis to analyze mass difference between two fractions.
  • To clarify the difference of molecular masses between Peak M and Peak V, peptide mapping analysis was carried out using UPLC/ESI-MS. First, a method for peptide mapping for rHu- EPO was developed for the UPLC with on-line Q-TOF system to check if glycopeptides having O-glycosylation can be detected.

대상 데이터

  • To identify deglycosylated peptides, the peptides were separated using an RP-UPLC equipped with an ACQUITY BEH300 C18 column (1.7 μm 2.1 x 150 mm).

이론/모형

  • 5. Fragmentation spectra of D16 peptide of Asp-N map (A) and E14 peptide of Glu-C map (B) from the fractions of RP-HPLC using the purity method. The precursor ion having 1103 m/z of Asp-N map and 1393 m/z of Glu-C map from the fraction of Peak M was fragmented and the structure of O-glycosylation (O-G1-2SA) was confirmed by the product ions.
  • 4. Identifcation of D16 peptide of Asp-N map (A) and E14 peptide of Glu-C map (B) on the MS1 spectrum of peptide map from the fractions of RP-HPLC using the purity method. Peak M represents the main peak fraction from Fig.
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참고문헌 (26)

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