Phosphoproteomics has become a popular proteomic technology for exploring cellular signaling networks. However, current approaches often require milligrams of protein samples which hamper their applications for translational studies with limited starting materials. In this study, we aimed to challenge the lowest starting material limit for phosphoproteome profiling. By carefully optimizing the well-established high-pH reversed-phase (RP) fractionation plus Ti4+-IMAC enrichment strategy, we achieved the identification of 15260 and 8936 unique phosphopeptides from only 500 g and 250 g predigested peptides, respectively. To further improve the sensitivity of phosphoproteome analysis for low micrograms of protein samples, we developed an integrated strategy, termed Phospho-SISPROT. This technology integrates three tips in tandem for protein digestion by the simple and integrated spintip-based proteomics technology (SISPROT), phosphopeptide enrichment by the Ti4+-IMAC tip, and desalting by the StageTip, respectively, which could dramatically reduce the phosphoproteome analysis time from a couple of days to only 6 hours and improve the system sensitivity. The flow through of Phospho-SISPROT could be reused for the global protein identification, which is very helpful for accurate phosphoproteome analysis with limited starting materials. More than 5500 and 600 unique phosphopeptides were respectively identified from 20 g and 1 g pervanadate treated HEK 293T cell lysates processed by the Phospho-SISPROT. To the best of our knowledge, this performance is the highest reported to date by using the standard LC-MS/MS setup. We expect that the Phospho-SISPROT and the optimized high-pH RP fractionation plus Ti4+-IMAC enrichment strategy will be well suited for highly sensitive phosphoproteome analysis of rare biological samples.
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Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Abelin, Jennifer G.
Trantham, Paisley D.
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Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Trantham, Paisley D.
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Penny, Sarah A.
Patterson, Andrea M.
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Univ Oklahoma, Dept Microbiol & Immunol, Oklahoma City, OK USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Patterson, Andrea M.
Ward, Stephen T.
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Univ Birmingham, Dept Clin Immunol, Birmingham, W Midlands, EnglandUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Ward, Stephen T.
Hildebrand, William H.
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Univ Oklahoma, Dept Microbiol & Immunol, Oklahoma City, OK USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Hildebrand, William H.
Cobbold, Mark
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Univ Birmingham, Dept Clin Immunol, Birmingham, W Midlands, EnglandUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Cobbold, Mark
Bai, Dina L.
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Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Bai, Dina L.
Shabanowitz, Jeffrey
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Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Shabanowitz, Jeffrey
Hunt, Donald F.
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Univ Virginia, Dept Chem, Charlottesville, VA 22903 USA
Univ Virginia, Dept Pathol, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
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Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Ficarro, Scott B.
Zhang, Yi
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Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Zhang, Yi
Carrasco-Alfonso, Marlene J.
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Harvard Univ, Sch Med, Brigham & Womens Hosp, Dept Pathol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Carrasco-Alfonso, Marlene J.
Garg, Brijesh
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Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Garg, Brijesh
Adelmant, Guillaume
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Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Adelmant, Guillaume
Webber, James T.
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Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Webber, James T.
Luckey, C. John
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Harvard Univ, Sch Med, Brigham & Womens Hosp, Dept Pathol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Luckey, C. John
Marto, Jarrod A.
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Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
机构:
Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Abelin, Jennifer G.
Trantham, Paisley D.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Trantham, Paisley D.
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Penny, Sarah A.
Patterson, Andrea M.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Oklahoma, Dept Microbiol & Immunol, Oklahoma City, OK USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Patterson, Andrea M.
Ward, Stephen T.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Birmingham, Dept Clin Immunol, Birmingham, W Midlands, EnglandUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Ward, Stephen T.
Hildebrand, William H.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Oklahoma, Dept Microbiol & Immunol, Oklahoma City, OK USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Hildebrand, William H.
Cobbold, Mark
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h-index: 0
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Univ Birmingham, Dept Clin Immunol, Birmingham, W Midlands, EnglandUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Cobbold, Mark
Bai, Dina L.
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h-index: 0
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Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Bai, Dina L.
Shabanowitz, Jeffrey
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h-index: 0
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Univ Virginia, Dept Chem, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
Shabanowitz, Jeffrey
Hunt, Donald F.
论文数: 0引用数: 0
h-index: 0
机构:
Univ Virginia, Dept Chem, Charlottesville, VA 22903 USA
Univ Virginia, Dept Pathol, Charlottesville, VA 22903 USAUniv Virginia, Dept Chem, Charlottesville, VA 22903 USA
机构:
Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Ficarro, Scott B.
Zhang, Yi
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h-index: 0
机构:
Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Zhang, Yi
Carrasco-Alfonso, Marlene J.
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h-index: 0
机构:
Harvard Univ, Sch Med, Brigham & Womens Hosp, Dept Pathol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Carrasco-Alfonso, Marlene J.
Garg, Brijesh
论文数: 0引用数: 0
h-index: 0
机构:
Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Garg, Brijesh
Adelmant, Guillaume
论文数: 0引用数: 0
h-index: 0
机构:
Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Adelmant, Guillaume
Webber, James T.
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h-index: 0
机构:
Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Webber, James T.
Luckey, C. John
论文数: 0引用数: 0
h-index: 0
机构:
Harvard Univ, Sch Med, Brigham & Womens Hosp, Dept Pathol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Luckey, C. John
Marto, Jarrod A.
论文数: 0引用数: 0
h-index: 0
机构:
Dana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA
Dana Farber Canc Inst, Blais Prote Ctr, Boston, MA 02115 USA
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USADana Farber Canc Inst, Dept Canc Biol, Boston, MA 02115 USA