Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins

Journal of Chromatography. a
E CalleriG Massolini

Abstract

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion...Continue Reading

References

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Citations

May 26, 2006·Analytical and Bioanalytical Chemistry·Lee Wah LimToyohide Takeuchi
Nov 29, 2012·Analytical and Bioanalytical Chemistry·Erika L PfaunmillerDavid S Hage
Jan 28, 2014·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Enrica CalleriGabriella Massolini
Sep 28, 2007·Analytical and Bioanalytical Chemistry·E C A StigterW P van Bennekom
Aug 12, 2009·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Kumiko Sakai-Kato, Keiko Ishikura
Aug 12, 2009·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·A CingözV Pichon
Mar 19, 2014·Analytical Chemistry·Nyoté J Oliver-CalixteSteven A Soper
Nov 23, 2006·Journal of Biochemical and Biophysical Methods·Shigenori OtaKazuki Nakanishi
Mar 21, 2016·Biochemical and Biophysical Research Communications·Guihua RuanFuyou Du
Feb 14, 2006·Protein Expression and Purification·M HedhammarS Hober
May 12, 2012·Journal of Analytical Methods in Chemistry·Supaporn Kradtap Hartwell, Kate Grudpan
Jun 8, 2012·Artificial Cells, Blood Substitutes, and Immobilization Biotechnology·Cenk Daglioglu, Figen Zihnioglu
Jun 17, 2016·Annual Review of Analytical Chemistry·Tohru Ikegami, Nobuo Tanaka
May 24, 2007·Molecular & Cellular Proteomics : MCP·Yun LiuBaohong Liu
Sep 15, 2006·Journal of Separation Science·Rangan Mallik, David S Hage
Mar 21, 2009·Journal of Separation Science·Caterina TemporiniGabriella Massolini
Jul 4, 2009·Journal of Separation Science·Wei Lin, Cameron D Skinner
Mar 3, 2010·Rapid Communications in Mass Spectrometry : RCM·Hyo-Jik YangJeongkwon Kim
Jul 13, 2006·Journal of Separation Science·John E SchielDavid S Hage
Jul 28, 2011·Journal of Separation Science·Jens Sproß, Andrea Sinz
Mar 16, 2013·Journal of Separation Science·Evgenia G Vlakh, Tatiana B Tennikova
Jan 8, 2013·Journal of Separation Science·Evgenia G Vlakh, Tatiana B Tennikova
Mar 17, 2017·Analytical and Bioanalytical Chemistry·Adam KecskemetiAttila Gaspar
Aug 28, 2013·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Atul Kumar Dwivedi, Parameswar Krishnan Iyer
Jul 31, 2009·Journal of Proteome Research·Hans W HahnGünther K Bonn
Feb 4, 2005·Journal of Separation Science·Gabriella Massolini, Enrica Calleri
Aug 12, 2021·Analytical and Bioanalytical Chemistry·Cynthia NagyAttila Gaspar

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