Ultra acidic strong cation exchange enabling the efficient enrichment of basic phosphopeptides.
We present a straightforward method to enrich phosphopeptides with multiple basic residues, an under-represented class in common enrichment strategies. Our method is based on a two-dimensional strong cation exchange (SCX) strategy, operating at two different acidic pHs, enabling both separation and enrichment of different classes of phosphopeptides. The principle of enrichment is based on the change of net charge of phosphorylated peptides under strong acidic conditions in the second SCX, whereas the net charge of regular peptides remains unchanged, thus enabling separation based on net charge. Application of our tandem SCX approach to a modest amount of human cells allowed the identification of over 10,000 unique "basic" phosphopeptides of which many represent putative targets of basophilic kinases.
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Marco L
ML
Hennrich
Henk W P
HW
van den Toorn
Vincent
V
Groenewold
Albert J R
AJ
Heck
Shabaz
S
Mohammed
D002412:Cations / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D002412:Cations; D010748:Phosphopeptides