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

Abstract

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 reversephase 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.

Rictor is a novel target of p70 S6 kinase-1

“…In some cases, definitive localization of the phosphorylation site could not be obtained (Figure 1a, sites marked in green). Some of these sites have previously been reported, including Ser21 and Ser1177, which have been identified in different largescale analyses of phosphoproteins (Beausoleil et al, 2004;Dai et al, 2007;Cantin et al, 2008;Daub et al, 2008;Dephoure et al, 2008;Zanivan et al, 2008). In addition, the phosphopeptides containing Ser1174, Ser1219, Thr1222, Thr1295, Ser1385, Tyr1386 and Ser1388, which have recently reported been, were also detected (Dephoure et al, 2008;Zanivan et al, 2008).…”

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

“…In parallel, in order to improve the annotation of the serovar Lai genome, ~1.37 million high-accuracy tandem mass spectrometry (MS/MS) spectra obtained by the Yin-yang multidimensional liquid chromatography (MDLC) system [27] coupled with LTQ-Orbitrap mass spectrometry (Yin-yang MDLC-MS/MS) from the serovar Lai were searched against the six-frame translation database. After filtering the identified peptides with a 1.0% false-positive ratio (FPR) [28], we selected those CDSs matched by at least two unique peptides to validate and rectify the genome annotation.…”

“…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.…”

Temporal and spatial profiling of nuclei-associated proteins upon TNF-α/NF-κB signaling

“…HUMAN.v3.28.REVERSED.fasta) using the TurboSEQUEST program. One missing trypsin cleavage site was allowed [51]. Carbamidomethylation was searched as a fixed modification and isotope-labeled lysine (+6.00 Da) was allowed as a variable modification [15].…”