Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder.
Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.
Feuerstein
Isabel
I
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria.
Morandell
Sandra
S
Stecher
Guenther
G
Huck
Christian W
CW
Stasyk
Taras
T
Huang
Hong-Lei
HL
Huber
Lukas A
LA
Bonn
Guenther K
GK
D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002482:Cellulose / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006736:Horses; D007098:Imino Acids / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011137:Polystyrenes / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
D002364:Caseins; D007098:Imino Acids; D008670:Metals; D009211:Myoglobin; D010748:Phosphopeptides; D011137:Polystyrenes; D019000:Phosphotyrosine; D002482:Cellulose; C003771:Amberlite XAD-2 resin; D019950:Mitogen-Activated Protein Kinase 1; C008109:iminodiacetic acid