Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform.
Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.
Weeks
Mark E
ME
Ludwig Institute for Cancer Research, Gower Street, London, UK. mark@ludwig.ucl.ac.uk
Sinclair
John
J
Jacob
Richard J
RJ
Saxton
Malcolm J
MJ
Kirby
Susan
S
Jones
James
J
Waterfield
Michael D
MD
Cramer
Rainer
R
Timms
John F
JF
D015180:Electrophoresis, Gel, Two-Dimensional; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D020543:Proteome / Q000378:metabolism*; D012371:Robotics; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020543:Proteome; D029702:Schizosaccharomyces pombe Proteins; D020928:Mitogen-Activated Protein Kinases; C095409:sty1 protein, S pombe