16. The Use of Peptide Training Kits to Build, Verify, and Evaluate Multiplexed Protein Quantitation Assays for Cross-Laboratory Studies
Mon 4:42 PM - PosterSplash Track 2
Scott Peterman
Thermo Fisher Scientific
Scott M. Peteman, Thermo Fisher Scientific, San Jose, CA
Amol Prakash, Thermo Fisher Scientific BRIMS, Cambridge, MA
Mary F. Lopez, Thermo Fisher Scientific BRIMS, Cambridge, MA
Taha Rezai, Thermo Fisher Scientific BRIMS, Cambridge, MA
John Rogers, Thermo Fisher Scientific Pierce, Rockford, Il
The drive to link proteomics and systems biology presents a new challenge to mass spectrometry. Large multiplexed assays designed to quantitate biological pathways and/or processes consist of a series of proteotypic peptides per targeted protein and a mass spectrometry-based method of quantifying the expression levels. Generally, researchers employ an SRM-based assay is used due to its inherent strengths but requires additional steps in method development to determine aspects needed for each SRM transition. To decrease the difficulties in building robust SRM assays designed for multiplexed quantitation, we utilize a set of well characterized set of heavy-labeled peptides. The trainer set is designed to characterize elution profiles, peak shapes, and cycle times needed to facilitate large SRM transition tables (>5000), verification, and QC for inter-laboratory studies.

A set of heavy labelled peptides were synthesize and delivered as an equal molar stock solution of 500 fmol/uL. (Thermo Fisher Scientific, Ulm, Germany) Yeast cell lysate samples were digested according to standard protocols and used at 500 ng on column. SRM assays were developed on a TSQ Quantum Ultra triple quadrupole mass spectrometer built from previously acquired shot-gun proteomics data acquired on an LTQ Orbitrap. Reverse phase separations were carried out on a 1mm X 250mm Hypersil Gold 3 ┬Ám C18 particle. Solvent A was LC-MS grade water with 0.2% (v/v) formic acid, and solvent B was LC-MS grade 30% (v/v) acetonitrile with 0.2% (v/v) formic acid. Four additional laboratories are used to analyze the same samples using different LC equipment. Pinpoint software was used for developing SRM assays. The software algorithm predicts candidate peptides and facilitates the choice of multiple fragment ions for SRM assay design, instrument method development, automatic peptide identity confirmation and quantitative data processing.

A complete workflow designed to perform multiplexed protein quantitation is presented. The workflow integrates a peptide trainer set to build, verify, and provide a QC analysis for an inter-laboratory study to demonstrate reproducibility on a common sample. All aspects of the experiment are controlled using Pinpoint to increase the robustness of the analysis and provide a means of data comparison. To demonstrate the effectiveness, a multiplexed assay is created for yeast, verified, and transferred to four outside laboratories for comparative analysis. Each laboratory utilizes a different LC set up for quantitation. Evaluations of inter-lab reproducibility will be presented for the peptide trainer kit as well as the targeted proteins in the yeast cell lysate digest.