Online Peptide Fractionation Using a Multiphasic Microfluidic Liquid Chromatography Chip Improves Reproducibility and Detection Limits for Quantitation in Discovery and Targeted Proteomics.
Christoph Krisp, Hao Yang, Remco van Soest, and Mark P Molloy
Molecular & Cellular Proteomics (2015) 14: 1708-1719.
This paper presents separation methods for improved reproducibility, which is often required for the workflows used in high-throughput comparative proteomics. The LC chip format enables fast workflow switching for easier transition from discovery to targeted studies, and provides a way to help standardize cross-laboratory proteomic analyses.
In the data-dependent MS/MS analysis of a cancer cell lysate 95% of all identified peptides were uniquely identified in a single fraction and less than 0.9% in more than two fractions. Using targeted detection with scheduled MRM high resolution MS, 503 peptides representing 173 plasma proteins were analyzed. In this multiphase fractionation approach all 503 peptides were quantifiable with a median CV of 3.4 +/- 3%, with only 10 peptides showing CVs > 15%. Three thyroid cell lines were analyzed with multiphase LC chip fractionation via data-independent MS/MS resulting in a total of 3875 proteins identified. You can access these data sets at ProteomeXchange.