Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography

Abstract

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoroteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combmation allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (∼0.7 μg) of bovine β-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.

Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS

“…That 2,5-DHB and CHCA are in fact able to solubilize mono-phosphopeptides from IMAC beads contradicts the majority of IMAC affinity column elution protocols, which exploit the use of high pH to disrupt the interaction between immobilized Fe 3ϩ and the bound phosphopeptides [2,8,16,17]. The pH of 2,5-DHB in a saturated aqueous solution is 2.0, whereas traditional The supernatant from (a) was removed, the beads were washed, and the remaining phosphopeptides eluted using 100 mM ammonium phosphate buffer, pH 4.6. IMAC protocols typically use the elution conditions of sodium phosphate at high pH (8 -10.5).…”

“…I mmobilized metal ion affinity chromatography (IMAC) has been used for a number of years for the selective enrichment of phosphopeptides from proteolytic digest mixtures containing both phosphorylated and non-phosphorylated components [1][2][3][4][5][6][7][8]. The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].…”

Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphoproteome and Improves Global Phosphopeptide Isolation and Detection

“…The most common strategy for enriching phosphopeptides in mixtures is the use of IMAC (10,11) or titanium dioxide (TiO 2 ) microcolumns (12,13). Although phosphopeptides have a very high affinity for these resins, acidic peptides (containing aspartic acid and glutamic acid) and peptides containing histidine also have a high binding affinity.…”

Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor

“…To selectively enrich the sample for phosphorylated peptides, immobilized metal affinity chromatography (IMAC) using Fe 3ϩ was used (10,25,29). This approach separates phosphopeptides from their non-phosphorylated counterparts and permits subsequent online (22,38) or offline (26) mass spectrometric analysis. In this study, offline IMAC enrichment used in conjunction with nHPLC-ESI MS permitted the identification of nine specific sites of phosphorylation in UBF.…”