Enhanced Detection of Multiply Phosphorylated Peptides and Identification of Their Sites of Modification

• Published in: Analytical chemistry (Washington) Vol. 85; no. 18; pp. 8566 - 8576
• Main Authors: Fleitz, Antoine, Nieves, Edward, Madrid-Aliste, Carlos, Fentress, Sarah J, Sibley, L. David, Weiss, Louis M, Angeletti, Ruth Hogue, Che, Fa-Yun
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.09.2013
• Subjects: Amino Acid Sequence; Analytical chemistry; Binding Sites - physiology; Cells; Cells, Cultured; Chromatography, Liquid - methods; Dynamic tests; Fibroblasts; Fibroblasts - chemistry; Fibroblasts - metabolism; Human; Humans; Male; Molecular Sequence Data; Peptides; Phosphopeptides - analysis; Phosphopeptides - genetics; Phosphopeptides - metabolism; Phosphorylation; Proteins; Strategy; Tandem Mass Spectrometry - methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac401691g

Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide–metal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Na2 or if 25 mM EDTA-Na2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of α-casein and ß-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either α- or ß-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the α-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 μg of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore ≥20). Of these sites 79% were mono-, 20% di-, and ∼1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.