Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry.
Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC-MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and Modiro(TM) v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.
Ahmed
Kareem Eldin A M
KE
Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, 1090, Vienna, Austria.
Chen
Wei-Qiang
WQ
John
Julius Paul Pradeep
JP
Kang
Sung Ung
SU
Lubec
Gert
G
D000588:Amines / Q000032:analysis / Q000737:chemistry; D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives / Q000032:analysis* / Q000737:chemistry; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D000069585:Filgrastim; D005973:Glutamine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008715:Methionine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D000588:Amines; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005973:Glutamine; C076022:5-(biotinamido)pentylamine; D016179:Granulocyte Colony-Stimulating Factor; D001710:Biotin; D008715:Methionine; D000069585:Filgrastim