title: Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis.
abstract: Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides 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 dimension 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 μg of cell lysate consumed. Our integrated RP-SAX-RP platform provides several analytical figures of merit, including: (1) orthogonal separation mechanisms in each dimension; (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 vitro model 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.
authors.affiliation: Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Marlene J
authors.initials: MJ
authors.lastname: Carrasco-Alfonso
authors.forename: Brijesh
authors.initials: B
authors.lastname: Garg
authors.forename: Guillaume
authors.initials: G
authors.lastname: Adelmant
authors.forename: James T
authors.initials: JT
authors.lastname: Webber
authors.forename: C John
authors.initials: CJ
authors.lastname: Luckey
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
mesh_terms: D056704:Adaptive Immunity; D000818:Animals; D057205:Automation, Laboratory; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange; D006801:Humans; D015470:Leukemia, Myeloid, Acute; D008088:Listeriosis / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D051941:fms-Like Tyrosine Kinase 3 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D010446:Peptide Fragments; D010750:Phosphoproteins; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3