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

Abstract:

: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.

journal_name

Amino Acids

journal_title

Amino acids

authors

Ahmed KE,Chen WQ,John JP,Kang SU,Lubec G

doi

10.1007/s00726-009-0312-1

subject

Has Abstract

pub_date

2010-04-01 00:00:00

pages

1043-9

issue

4

eissn

0939-4451

issn

1438-2199

journal_volume

38

pub_type

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