Protein Phosphorylation and Expression Profiling by Yin-Yang Multidimensional Liquid Chromatography (Yin-Yang MDLC) Mass Spectrometry

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with
mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange
(SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX
column. The flow-through peptides from SCX were collected and further loaded on an SAX column.
Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was
achieved by the Yin-yang MDLC−MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were
identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with
809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features
of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass
spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated
peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134
groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but
different phosphorylated states. This unbiased profiling of protein expression and phosphorylation
provides a powerful approach to probe protein dynamics, without using any prefractionation and
chemical derivation.
Keywords: Protein phosphorylation • Protein expression • Strong-cation exchange • Strong-anion exchange • Yin-Yang multidimensional liquid chromatography • pH elution • Mass spectrometry