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.

Comparative studies of peak intensities and chromatographic separation of proteolytic digests, PTMs, and intact proteins obtained by nanoLC-ESI MS analysis at room and elevated temperatures

“…In addition, the ion suppression effect detrimental for attaining high sensitivity in the LC-MS analysis of complex mixtures was shown to be less pronounced at flow rates below 20 nL/min when the LC solvents were electrosprayed using 2-3 μm i.d. emitters [57,58]. Our data demonstrate that the ion suppression still exists even at very low flow rates but the suppression effect can be further reduced by heating both the LC column and ESI tip to high temperatures.…”

Rapid Preconcentration for Liquid Chromatography–Mass Spectrometry Assay of Trace Level Neuropeptides

“…The dual pump system with selection valve allows sample to be loaded onto the column at high flow rate but then eluted at a lower flow rate that gives better ESI sensitivity [40,41] and LC performance without requiring an extensive pressure equilibration [26]. Despite these steps, the rate limiting step of analysis is preconcentration and rinsing [17,26,27].…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…A commercially available sheathless CESI source combines high resolution separation of CE with high resolution mass spectrometry. Because there is no sample dilution, as occurs in sheath-flow systems, the sheathless interface is more sensitive [235][236][237]. We [238] and others [229,239] have demonstrated the potential of high resolution CZE coupled to high resolution MS to analyze intact complex glycoproteins.…”