Mass spectrometry (MS) has become the instrument of choice for direct identification of peptides. Superior to traditional immunological techniques, such as RIA, ELISA or IHC, MS enables simultaneous large-scale analysis of peptides and avoids the discrepancies associated with inconsistent antibody affinities.
The Proteomics Core Facility (PCF) at the University of Gothenburg, in collaboration with scientists from Denator AB, has established a protocol for large-scale analysis of endogenous peptides from heat-stabilized tissue samples using high resolution, nano LC-MS to provide simultaneous identification and relative quantification.
This advanced workflow will facilitate the global analysis of endogenous peptides and be particularly useful when comparing control/diseased/treated or heat-stabilized/non-stabilized samples.
PCF is pleased to offer this service to enable many laboratories to perform global comparisons of peptide samples.
Frozen tissue samples are sent to PCF together with a sample submission form describing experimental parameters such as ex-vivo time, number of samples, project scope etc. Each frozen sample is then heat stabilized using the Stabilizor™ system (Denator AB) to prevent degradation of peptides and proteins that would mask the presence of endogenous peptides. The heat-stabilized tissue is homogenized in a water-based, denaturation buffer, including an ultrasonication step to facilitate maximal peptide extraction. Proteins are removed by size exclusion. The peptide fraction is analyzed by nano LC-MS to determine peptide mass and relative quantification (MS) as well as peptide sequence information after peptide fragmentation (MS/MS). Combining these data enables identification of most peptides.
Data are analyzed using computer software such as Mascot™ (Matrix Science) for peptide identification and Progenesis™ (Nonlinear Dynamics) for relative quantification of detected peptides between all samples. Results are delivered to the customer as raw data in an Excel file and as a pdf report.
Learn more about:
Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome. Birger Scholz, Karl Sköld, Kim Kultima, Celine Fernandez, Sofia Waldemarson, Mikhail M. Savitski, Marcus Söderquist, Mats Borén, Robert Stella, Per Andrén, Roman Zubarev and Peter James
Mol Cell Proteomics. 2011 Mar;10(3):M900229MCP200. Epub 2010 Jan 28
Neuropeptide profiling of the bovine hypothalamus: Thermal stabilization is an effective tool in inhibiting post-mortem degradation Michelle L. Colgrave, Li Xi, Sigrid A. Lehnert, Traute Flatscher-Bader, Henrik Wadensten, Anna Nilsson, Per E. Andren, Gene Wijffels
PROTEOMICS Volume 11, Issue 7, pages 1264–1276, April 2011
Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics Marcus Svensson, Mats Borén, Karl Sköld, Maria Falth, Benita Sjögren, Malin Andersson, Per Svenningsson and Per E. Andrén
J Proteome Res. 2009 Feb;8(2):974-81
MS the Discovery of Novel Peptides from the Brain Marcus Svensson, Karl Sköld, Anna Nilsson, Maria Fälth Katarina Nydahl, Per Svenningsson, Per E. Andrén
Anal Chem January 1, 2007 Volume 79, Issue 1, pp14-21
The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin 2-20 and peptides as sample quality indicators Karl Sköld, Marcus Svensson, Mathias Norrman, Benita Sjögren, Per Svenningsson, Per E. Andrén
PROTEOMICS Volume 7, Issue 24, pages 4445–4456, No. 12 December 2007