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Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology.
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2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 19Article in journal (Refereed) Published
Abstract [en]

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.

Place, publisher, year, edition, pages
2004. Vol. 76, no 19
URN: urn:nbn:se:umu:diva-110771DOI: 10.1021/ac040044gPubMedID: 15456308OAI: diva2:865370
Available from: 2015-10-28 Created: 2015-10-28 Last updated: 2015-10-28

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