Automated serum peptide profiling using novel magnetic C18 beads off-line coupled to MALDI-TOF-MS.
Serum peptide profiling by MS is an emerging approach for disease diagnosis and biomarker discovery. A magnetic bead-based method for off-line serum peptide capture coupled to MALDI-TOF-MS has been recently introduced. However, the reagents are not available to the general scientific community. Here, we developed a protocol for serum peptide capture using novel magnetic C18 beads, and automated the procedure on a high-throughput magnetic particle processor. We investigated bead equilibration, peptide binding and peptide elution conditions. The method is evaluated in terms of peaks counts and reproducibility of ion intensities in control serum. Overall, the DynaBead-RPC18-based serum sample processing protocol reported here is reproducible, robust and allows for the detection of ˜200 peptides at m/z 800-4000 of serum that was allowed to clot for 1 h. The average intra-experiment %CV of normalized ion intensities for crude serum and 0.5% TFA/0.15% n-octyl glucoside-treated serum, respectively, were 12% (range 2-38%) and 10% (3-21%) and the inter-experiment %CVs were 24% (10-53%) and 31% (10-59%). Importantly, this method can be used for serum peptide profiling by anyone in possession of a MALDI-TOF instrument. In conjunction with the KingFisher® 96, the whole serum peptide capture procedure is high-throughput (˜20 min per isolation of 96 samples in parallel), thereby facilitating large-scale disease profiling studies.
OncoProteomics Laboratory, Department of Medical Oncology, VU Medical Center, Amsterdam, The Netherlands; Department of Molecular and Cellular Neurobiology, Center for Medical Systems Biology/Neurogenomics and Cognitive Research, Vrije Universteit, Amsterdam, The Netherlands. c.jimenez@vumc.nl.
Connie R
CR
Jimenez
Zineb
Z
El Filali
Jaco C
JC
Knol
Klaas
K
Hoekman
Frank A E
FA
Kruyt
Giuseppe
G
Giaccone
August B
AB
Smit
Ka Wan
KW
Li