NDLI: Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS

Content Provider	Springer Nature Link
Author	Seidler, Joerg Zinn, Nico Haaf, Erik Boehm, Martin E. Winter, Dominic Schlosser, Andreas Lehmann, Wolf D.
Copyright Year	2010
Abstract	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 μLC-ICP-MS or nanoLC-ESI-MS. Using μLC-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 Ga3+-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.
Starting Page	311
Ending Page	320
Page Count	10
File Format	PDF
ISSN	09394451
Journal	Amino Acids
Volume Number	41
Issue Number	2
e-ISSN	14382199
Language	English
Publisher	Springer Vienna
Publisher Date	2010-06-16
Publisher Place	Vienna
Access Restriction	Subscribed
Subject Keyword	Phosphopeptides Metal ion mobilization Metal ion chelators Metal-free LC Phosphopeptide recovery Multiply phosphorylated peptides Analytical Chemistry Life Sciences Neurobiology Biochemistry Proteomics Biochemical Engineering
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Biochemistry Clinical Biochemistry

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