Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification

Antoine Fleitz, Edward Nieves, Carlos Madrid-Aliste, Sarah J. Fentress, L. David Sibley, Louis M. Weiss, Ruth Hogue Angeletti, Fa Yun Che

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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-Na 2 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.

Original languageEnglish (US)
Pages (from-to)8566-8576
Number of pages11
JournalAnalytical Chemistry
Volume85
Issue number18
DOIs
StatePublished - Sep 17 2013

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Edetic Acid
Phosphopeptides
Caseins
Peptides
Phosphorylation
Phosphoproteins
Fibroblasts
Metal ions
Cells
Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification. / Fleitz, Antoine; Nieves, Edward; Madrid-Aliste, Carlos; Fentress, Sarah J.; Sibley, L. David; Weiss, Louis M.; Angeletti, Ruth Hogue; Che, Fa Yun.

In: Analytical Chemistry, Vol. 85, No. 18, 17.09.2013, p. 8566-8576.

Research output: Contribution to journalArticle

Fleitz, A, Nieves, E, Madrid-Aliste, C, Fentress, SJ, Sibley, LD, Weiss, LM, Angeletti, RH & Che, FY 2013, 'Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification', Analytical Chemistry, vol. 85, no. 18, pp. 8566-8576. https://doi.org/10.1021/ac401691g
Fleitz, Antoine ; Nieves, Edward ; Madrid-Aliste, Carlos ; Fentress, Sarah J. ; Sibley, L. David ; Weiss, Louis M. ; Angeletti, Ruth Hogue ; Che, Fa Yun. / Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification. In: Analytical Chemistry. 2013 ; Vol. 85, No. 18. pp. 8566-8576.
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abstract = "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-Na 2 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.",
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AU - Sibley, L. David

AU - Weiss, Louis M.

AU - Angeletti, Ruth Hogue

AU - Che, Fa Yun

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