Journal of Chromatography B, 817 (2005) 81–88 Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme Erin H. Seeley, Larry D. Riggs, Fred E. Regnier ∗ Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA Available online 13 April 2004 Abstract The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples. © 2004 Elsevier B.V. All rights reserved. Keywords: Phosphopeptides; Endoproteinase glu-C 1. Introduction Phosphorylation is one of the more abundant posttransla- tional modifications (PTM) of proteins, being implicated in cellular functions ranging from signal transduction and gene expression to the regulation of growth and involvement in disease mechanisms. Accordingly, much effort has been ex- pended in the study and quantification of changes in protein phosphorylation and dephosphorylation [1–14]. One of the oldest methods for measuring phosphoryla- tion is through 32 P labeling with quantification by scintil- lation counting or audioradiography [1,2]. More recently, quantification is achieved by stable isotope labeling and mass spectrometry. Because phosphoproteins appear in low abundance, some type of enrichment is often used to obtain sufficient quantities of material for characterization. Anti- bodies have been relatively useful in the context of recog- nition and enrichment, particularly with Western blotting and immunoprecipitation methods associated with 2D gel ∗ Corresponding author. Tel.: +1-765-494-3878; fax: +1-765-494-0359. E-mail address: fregnier@purdue.edu (F.E. Regnier). electrophoresis [3,4]. They are fairly specific for the 0.1% of phosphoproteins that are phosphorylated on tyrosine [5] but seem to be far less useful in the enrichment of phos- phoserine and phosphothreonine containing proteins [4]. At the peptide level of phosphoprotein analysis, a method for the replacement of the phosphate ester with another func- tional group carrying an affinity selectable tag has been de- scribed [5–8]. This approach exploits the fact that phosphate esters on serine and threonine -eliminate with the forma- tion of an ,-unsaturated double bond. The -elimination product is then alkylated at the former phosphorylation site with ethane dithiol (EDT) and subsequently labeled with a biotin tag. Following the removal of excess EDT-biotin tag- ging agent, biotinylated peptides are selected with an avidin column and then characterized by LC/MS. Phosphotyrosine does not -eliminate, making this method specific for serine and threonine phosphorylated peptides. Because serine and threonine phosphorylation sites tend to cluster, it is com- mon to encounter multiple biotin groups in the same pep- tide when using this process. Biotinylation adds roughly 500 amu per phosphorylation site to the mass of a peptide, caus- ing the mass of peptides with multiple phosphorylation sites to be substantially higher after biotin tagging. It is impor- 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.03.024