title: Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics.
abstract: The potential benefits of ultra-low flow electrospray ionization (ESI) for the analysis of phosphopeptides in proteomics was investigated. First, the relative flow dependent ionization efficiency of nonphosphorylated vs multiplyphosphorylated peptides was characterized by infusion of a five synthetic peptide mix with zero to four phophorylation sites at flow rates ranging from 4.5 to 500 nL/min. Most importantly, similar to what was found earlier by Schmidt et al., it has been verified that at flow rates below 20 nL/min the relative peak intensities for the various peptides show a trend toward an equimolar response, which would be highly beneficial in phosphoproteomic analysis. As the technology to achieve liquid chromatography separation at flow rates below 20 nL/min is not readily available, a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) strategy based on the use of a neutrally coated separation capillary was used to develop an analytical strategy at flow rates as low as 6.6 nL/min. An in-line preconcentration technique, namely, transient isotachophoresis (t-ITP), to achieve efficient separation while using larger volume injections (37% of capillary thus 250 nL) was incorporated to achieve even greater sample concentration sensitivities. The developed t-ITP-ESI-MS strategy was then used in a direct comparison with nano-LC-MS for the detection of phosphopeptides. The comparison showed significantly improved phosphopeptide sensitivity in equal sample load and equal sample concentration conditions for CE-MS while providing complementary data to LC-MS, demonstrating the potential of ultra-low flow ESI for the analysis of phosphopeptides in liquid based separation techniques.
authors.affiliation: Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands. a.a.m.heemskerk@lumc.nl
authors.forename: Anthonius A M
authors.initials: AA
authors.lastname: Heemskerk
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Busnel
authors.forename: Bart
authors.initials: B
authors.lastname: Schoenmaker
authors.forename: Rico J E
authors.initials: RJ
authors.lastname: Derks
authors.forename: Oleg
authors.initials: O
authors.lastname: Klychnikov
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Oleg A
authors.initials: OA
authors.lastname: Mayboroda
mesh_terms: D000818:Animals; D057625:Isotachophoresis; D008892:Milk / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D010748:Phosphopeptides