Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry

Abstract

Most current technologies for the enrichment of phosphopeptides rely on a tandem combination of different chromatography modes. Here, a fully automatic two-dimensional liquid chromatography mass spectrometry method was developed for global phosphopeptide identification. The peptide mixtures were loaded on a strong anion exchange (SAX) column under basic pH conditions and eluted with a continuous gradient to pH 2.0. This SAX system could be coupled online with reversed-phase liquid chromatography mass spectrometry (RP-LC-MS/MS). For peptide digests from a standard protein mixture spiked with synthesized phosphopeptides, most of the nonphosphorylated peptides were eluted in more basic pH than phosphopeptides, and the phosphopeptides were focused to acidic pH ranges and gradually eluted according to the phosphorylated states of peptides. Compared with the pH step elution method, the continuous gradient method displayed better repeatability and less peptide cross-overlap between fractions. This system provided a robust and fully automatic approach to large-scale phosphoproteomic profiling. For protein tryptic digests from HeLa cells, 1833 nonredundant phosphorylation sites were identified based on this two-phase separation. Compared with the method combining cation exchange and titanium dioxide, this anion-exchange based system preferred to identify more acidic and multiphosphorylated peptides. It also covered a more complete series of phosphorylation states of peptides.

High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans

“…The Yin-yang MDLC system was performed as described previously with some modifications including using pH continuous gradient elution instead of pH step gradient elution and using SAX as the first loading [27,47]. Briefly, ~1 mg of the sample was dissolved in 100 μl of pH 2.0 buffer (2 mM citric acid adjusted by formic acid), and then loaded onto the SCX column (10 μm, 320 μm × 100 mm, Column Technology Inc, CA, USA) by a syringe pump at a flow rate of 3 μl/min.…”

“…The HPLC solvents used were 0.1% formic acid (v/v) aqueous (A) and 0.1% formic acid (v/v) acetonitrile (ACN) (B). The sequential elution from the SCX column was by pH continuous gradient buffer, which was from pH 2.0 to pH 8.5 (from pH 8.5 to pH 2.0 in SAX) instead of previously reported pH step gradient buffer [27], as described in our recent work [47]. Each of the 10 eluted fractions was on-line concentrated and desalted on the C 18 trap column at a flow rate of 3 μl/min after the split, and then subjected to the analytical C18 column.…”

Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

“…Examples of first-dimension separations include hydrophilic interaction liquid chromatography (28), electrostatic repulsion hydrophilic interaction chromatography (29), strong anion exchange (30), and strong cation exchange (11,31). Although powerful and frequently used, enrichment after fractionation can be irreproducible and time consuming.…”

Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

“…To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4]. To reduce the interference of non-phosphopeptides, selective enrichment strategies, such as immobilized metal ion affinity chromatography (IMAC) [5][6][7], metal oxide affinity chromatography (MOAC) [8,9], and ion exchange chromatography [10][11][12][13], are necessary before MS analysis.…”

“…To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4]. To reduce the interference of non-phosphopeptides, selective enrichment strategies, such as immobilized metal ion affinity chromatography (IMAC) [5][6][7], metal oxide affinity chromatography (MOAC) [8,9], and ion exchange chromatography [10][11][12][13], are necessary before MS analysis.Recently, chip-based system has been introduced as a promising platform for phosphopeptide analysis, due to the benefits of low sample amount, high throughput, and easy integration [14]. Several microfluidic devices have been developed by integrating phosphopeptide enrichment with other functions, such as protein alkylation and reduction [15], protein digestion [16], peptide separation and on-line interface for MS instrument [17][18][19][20], which have shown great potentials in the field of phosphoproteome research.…”