Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry

• Published in: Amino acids Vol. 38; no. 4; pp. 1043 - 1049
• Main Authors: Ahmed, Kareem Eldin A. M, Chen, Wei-Qiang, John, Julius Paul Pradeep, Kang, Sung Ung, Lubec, Gert
• Format: Journal Article
• Language: English
• Published: Vienna Vienna : Springer Vienna 01.04.2010; Springer Vienna; Springer Nature B.V
• Subjects: Amines - analysis; Amines - chemistry; Amino Acid Sequence; Analytical Chemistry; Biochemical Engineering; Biochemistry; Biomedical and Life Sciences; Biotin - analogs & derivatives; Biotin - analysis; Biotin - chemistry; Biotinylation; Chromatography, High Pressure Liquid; Computer programs; Electrophoresis, Gel, Two-Dimensional; Enzymes; Filgrastim; Glutamine - analogs & derivatives; Glutamine - analysis; Glutamine - chemistry; Granulocyte Colony-Stimulating Factor - chemistry; Granulocyte Colony-Stimulating Factor - isolation & purification; Granulocyte Colony-Stimulating Factor - metabolism; Humans; Life Sciences; Mass spectrometry; Medical research; Methionine; Methionine - analogs & derivatives; Methionine - analysis; Methionine - chemistry; Microchemistry - methods; Molecular Sequence Data; Molecular Structure; Molecular Weight; Nanostructure; Neurobiology; Original Article; Oxidation-Reduction; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptides; Proteins; Proteomics; Recombinant; Recombinant Proteins - chemistry; Sequence Analysis, Protein - methods; Sequencing; Software; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Mascot; Two-dimensional electrophoresis; Modiro; Filgrastim; PentylamineBiotin; Complete sequencing
• DOI: 10.1007/s00726-009-0312-1
• ISSN: 0939-4451

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 ModiroTM 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.