[Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry].
The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.
Yu
Rong
R
Key Laboratory of Drug Targeting and Drug Delivery System of Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu 610041, China. yurong@scu.edu.cn
Zhang
Gui-feng
GF
Gao
Ling
L
Su
Zhi-guo
ZG
Wu
Wu-tong
WT
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D002918:Chymotrypsin / Q000737:chemistry; D005343:Fibrinolytic Agents / Q000032:analysis / Q000737:chemistry; D006629:Hirudins / Q000032:analysis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D010959:Tissue Plasminogen Activator / Q000032:analysis* / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
D005343:Fibrinolytic Agents; D006629:Hirudins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C087896:reteplase; D002918:Chymotrypsin; D014357:Trypsin; D010959:Tissue Plasminogen Activator