Immobilized Metal Affinity Chromatography Revisited: pH/Acid Control toward High Selectivity in Phosphoproteomics

Abstract

Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, a highly specific enrichment protocol is still a challenge in large-scale studies. Here, we report a simple pH/acid control method that addresses the poor specificity seriously criticized in IMAC. Detailed evaluation of the capture and release mechanism in IMAC revealed that pH, buffer and salt yield a complex interplay in enrichment of phosphopeptides, yet they play individual roles in recovery and specificity. A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the structure and concentration of organic acid. Without methyl esterification, the one-step IMAC enrichment with single LC-MS/MS identified 386 phosphoproteins in 550 mug of non-small-cell lung cancer cell lysate with 96% specificity. Additional fractionation by SDS-PAGE from 4 mg of cell lysate revealed the comprehensive proteome map, identifying 2747 phosphorylation sites from 2360 nondegenerate phosphopeptides and 1219 phosphoproteins with a false discovery rate of 0.63%. To our knowledge, this pH/acid-controlled IMAC procedure provides higher specificity than any other one-step IMAC purification procedure. Furthermore, the simple and reproducible IMAC protocol can be adapted to other solid supports, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome.

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

“…We compiled data from 13 independent proteomic studies (Amanchy et al, 2005;Brill et al, 2004;Cantin et al, 2008;Daub et al, 2008;Guo et al, 2008;Olsen et al, 2006;Pan et al, 2008;Rikova et al, 2007;Rush et al, 2005;Schlosser et al, 2006;Sui et al, 2008;Tao et al, 2005;Tsai et al, 2008), which identified 13 phosphorylated tyrosines in emerin (Fig. 1A).…”

Strategies for Protein Separation

“…They characterized over 2,360 nondegenerate phosphopeptides and 2,747 phosphorylation sites in the H1299 lung cancer cell line, showing that a low pH buffer increases the specificity of IMAC for phosphopeptides. The buffer composition was directly associated to the specificity and selectivity of the IMAC technique (Tsai et al, 2008;Jensen et al, 2007). Another important enrichment technique widely used is the metal oxide affinity chromatography (MOAC).…”

Linking the proteins—Elucidation of proteome‐scale networks using mass spectrometry

“…Briefly, IMAC with Fe (III) or Ga (III) has been widely used to purify phosphopeptides, even though co-selection of non-modified sequences has been described as a major pitfall of the technique. Esterification of peptide carboxyl groups provides an increased specificity of phosphopeptide enrichment (Ficarro et al, 2002); alternatively, pH, buffer, and salt conditions have been tested to optimize the IMAC procedure (Tsai et al, 2008a). A double use of IMAC might be performed, at the protein and peptide levels, to increase phosphopeptide purification specificity (Cantin et al, 2008), and a fractionation step has also been widely performed before or after phosphopeptide enrichment on IMAC material: strong anion exchange (SAX) (Nuhse et al, 2004), SCX , hydrophilic interaction chromatography (HILIC) (Albuquerque et al, 2008;McNulty & Annan, 2008), Poros resins or graphite powder columns (Boeri Erba et al, 2005).…”