Improved enrichment and proteomic analysis of brain proteins with signaling function by heparin chromatography.
Detection of low-abundance proteins with signaling function is essential for the identification of biomarkers and novel drug targets. We present a protocol for specific enrichment of secreted proteins with signaling function by combining subcellular fractionation with heparin chromatography. The subcellular fractionation includes the preparation of a fraction enriched in cytosolic proteins. A further enrichment was achieved by heparin chromatography. The proteins eluted from the heparin column were analyzed by MudPIT tandem mass spectrometry and identified with the use of an in silico algorithm. Forty-eight percent of the identified proteins (188 out of 391) bound to the heparin matrix. Fifty-four percent of them (101) are secreted proteins with signaling function and 23% (44) of the enriched signaling proteins had not been detected by 2D PAGE without application of the heparin enrichment step. The heparin chromatography method can be combined with other proteomics enrichment approaches, such as ion exchange or reversed phase chromatography.
Krapfenbauer
Kurt
K
Novartis Institutes for Biomedical Research, Vienna, Austria. kurt.krapfenbauer@novartis.com
Fountoulakis
Michael
M
D000818:Animals; D001923:Brain Chemistry; D002845:Chromatography / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006493:Heparin / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D053719:Tandem Mass Spectrometry / Q000379:methods
D009419:Nerve Tissue Proteins; D020543:Proteome; D006493:Heparin