Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis

Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phos-phoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phos-phopeptide enrichment. Here we explore two novel mul-tidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize ali-phatic ion pairing agents to improve retention of phos-phopeptides at high pH in the first dimension of a two-dimensional RP-RP. The second approach is based on the addition of strong anion exchange as the second dimen-sion in a three-dimensional reversed phase (RP)-strong anion exchange (SAX)-RP configuration. Both techniques provide for automated, online data acquisition, with the 3-D platform providing the highest performance both in terms of separation peak capacity and the number of unique phosphopeptide sequences identified per g of cell lysate consumed. Our integrated RP-SAX-RP plat-form provides several analytical figures of merit, includ-ing: (1) orthogonal separation mechanisms in each dimen-sion; (2) high separation peak capacity (3) efficient retention of singly- and multiply-phosphorylated peptides; (4) compatibility with automated, online LC-MS analysis. We demonstrate the reproducibility of RP-SAX-RP and apply it to the analysis of phosphopeptides derived from multiple biological contexts, including an in vitromodel of acute myeloid leukemia in addition to primary polyclonal CD8 T-cells activated in vivo through bacterial infection and then purified from a single mouse. Molecular &amp