Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS.
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Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS. / Seidler, Joerg; Zinn, Nico; Haaf, Erik; Boehm, Martin E; Winter, Dominic; Schlosser, Andreas; Lehmann, Wolfgang.
in: AMINO ACIDS, Jahrgang 41, Nr. 2, 2, 2011, S. 311-320.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS.
AU - Seidler, Joerg
AU - Zinn, Nico
AU - Haaf, Erik
AU - Boehm, Martin E
AU - Winter, Dominic
AU - Schlosser, Andreas
AU - Lehmann, Wolfgang
PY - 2011
Y1 - 2011
N2 - It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase ?LC-ICP-MS or nanoLC-ESI-MS. Using ?LC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.
AB - It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase ?LC-ICP-MS or nanoLC-ESI-MS. Using ?LC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.
KW - Reference Standards
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Adsorption
KW - Aluminum/chemistry
KW - Ascorbic Acid/chemistry
KW - Chromatography, Reverse-Phase/instrumentation/methods/standards
KW - Coordination Complexes/chemistry
KW - Deferoxamine/chemistry
KW - Imidazoles/chemistry
KW - Iron/chemistry
KW - Nanotechnology/methods/standards
KW - Peptide Fragments/chemistry/standards
KW - Phosphoproteins/chemistry/standards
KW - Phosphorus/chemistry
KW - Titanium/chemistry
KW - Reference Standards
KW - Amino Acid Sequence
KW - Molecular Sequence Data
KW - Adsorption
KW - Aluminum/chemistry
KW - Ascorbic Acid/chemistry
KW - Chromatography, Reverse-Phase/instrumentation/methods/standards
KW - Coordination Complexes/chemistry
KW - Deferoxamine/chemistry
KW - Imidazoles/chemistry
KW - Iron/chemistry
KW - Nanotechnology/methods/standards
KW - Peptide Fragments/chemistry/standards
KW - Phosphoproteins/chemistry/standards
KW - Phosphorus/chemistry
KW - Titanium/chemistry
M3 - SCORING: Journal article
VL - 41
SP - 311
EP - 320
JO - AMINO ACIDS
JF - AMINO ACIDS
SN - 0939-4451
IS - 2
M1 - 2
ER -
