Agilent AssayMAP Bravo Technology Enables Reproducible Automated Phosphopeptide Enrichment from Complex Mixtures Using High‐Capacity Fe(III)‐NTA Cartridges

Immobilized metal affinity chromatography (IMAC) using a nitrilotriacetic acid (NTA) chelating ligand functionalized with Fe(III) is regarded as one of the most selective phosphopeptide enrichment techniques for LC/MS applications. However, the use of IMAC for phosphopeptide enrichment often results in unacceptably high sample‐to‐sample variability. Phosphopeptide enrichment is reliant on manual sample preparation methods resulting in workflows lacking robustness except in the hands of highly trained scientists. Recently, several protein and peptide sample preparation workflows were automated using the Agilent AssayMAP Bravo platform, which reduced sample preparation variability, provided a clear path for sample processing scalability, and enabled customized workflow development without the need for in‐house automation expertise. We now report on the use of high‐capacity Agilent AssayMAP Fe(III)‐NTA cartridges for automated phosphopeptide enrichment featuring the AssayMAP Bravo. We describe the capacity of AssayMAP Fe(III)‐NTA cartridges, explain chemistry considerations for successful phosphopeptide enrichment by IMAC, and demonstrate that thousands of phosphopeptides can be reproducibly purified from yeast tryptic digests with high specificity in an automated fashion facilitated by high‐resolution LC/MS.