Human breast cancer cell line phosphoproteome revealed by an automated and highly selective enrichment workflow

The Agilent AssayMAP Bravo platform provides a fully automated, highly selective, and reproducible enrichment workflow using Fe(III)-NTA cartridges. To evaluate how the ratio of total peptide sample amount to affinity resin affects the performance and reproducibility of phosphopeptide enrichment and monitor the yield and reproducibility of the enrichment using phosphopeptide standards, Agilent joined the part II initiative proposed by the European Proteomics Association (EuPA).

The samples consisted of a tryptic digest of the human MCF7 breast cancer cell line (C-18 purified), spiked with a mixture of 20 human phosphopeptide standards containing light isotopes. The cell line digest samples were loaded onto the cartridges with four different sample/resin ratios.

The enrichment result showed excellent selectivity (>90 %) across all samples with different sample/resin ratios.

Phosphopeptide selectivity increased from 1.5 % before enrichment to over 90 % after enrichment for all samples. AssayMAP Bravo enabled reproducible enrichment resulting in consistent high selectivity for all samples. Across the four sample/resin ratios, we found that the distinct number of phosphopeptides increased with more sample loading, but not proportional to the ratio.

The yield for each phosphopeptide within the same sample varied, ranging from less than 10 % to nearly 100 %. However, the same phosphopeptide standards showed consistent yield across different sample/resin ratios, with approximately 63 % overall recovery for the 20 phosphopeptide standards. The yield for each phosphopeptide standard showed excellent reproducibility, with less than 5 % RSD of the enrichment and robustness of the peptide quantitation.