Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis

Abstract

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectrometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotein starting material. Miniaturized sample preparation methods for MS facilitated localization of phosphorylation sites in phosphoproteins isolated by polyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(III) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichment of phosphorylated peptides from crude peptide mixtures prior to off-line analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nanoelectrospray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was implemented, which in combination with nano-scale Fe(III)-IMAC and MALDI-MS allowed unambiguous identification of phosphopeptides by observation of 80 Da mass shifts. Nanoelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine beta-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site at Ser-3 in recombinant human casein kinase-2 beta subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein isolated by two-dimensional gel electrophoresis.

Selective Depletion and Enrichment Methods for the Analysis of Protein and Peptide Pools

“…Phosphate groups show a selective complexation affinity for transition metal ions and this can be used to collect phosphopeptides on an immobilized metal affinity column (IMAC) from which they can be subsequently eluted [62]. In the original form, IMAC columns consisted of iminodiacetate-or nitrilotriacetate--bearing materials complexed with Fe 3+ , Ga 3+ , or other metals [63][64][65][66]. Phosphopeptides are selectively retained under acidic conditions (pH 2.5-3.5) and eluted with alkali (approximately pH 10).…”

Identification of protein phosphorylation sites by combination of elastase digestion, immobilized metal affinity chromatography, and quadrupole-time of flight tandem mass spectrometry

“…The relatively small size of phosphopeptides produced by elastase is better suited for MS/MS than the generally larger tryptic phosphopeptides, the product ion spectra of which often show little sequence information. Moreover, large size phosphopeptides show a reduced recovery in IMAC enrichment [26]. Therefore, in this study on protein phosphorylation analysis, we again selected digestion by elastase, and combined this method with IMAC enrichment to reduce the complexity of the elastase digest to a point where complete characterization by automated nanoESI MS/MS becomes feasible.…”

“…In addition, pSer and pThr peptides have been detected by scanning for neutral loss of H 3 PO 4 [19][20][21]. Alternatively phosphopeptides can be selectively enriched by IMAC [22][23][24][25][26], a step which considerably reduces the complexity of the peptide mixture subjected to mass spectrometric analysis. Recently, two novel strategies for proteome-wide analysis of protein phosphorylation were introduced, based on covalent modification with biotin at the site of phosphorylation and subsequent isolation of biotinylated peptides by affinity chromatography [27,28].…”

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization- time of flight-mass spectrometry: II. Biological applications

“…The digested samples were pretreated by microextraction followed by either on-target sample deposition or direct microdispensing. Dephosphorylation of phosphopeptides can be accomplished by treatment with alkaline phospatase [32,33].…”