Detection of Phosphorylated Peptides in Proteomic Analyses Using Microfluidic Compact Disk Technology
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- 作者：Daniel Hirschberg ; Theres Jä ; gerbrink ; Jenny Samskog ; Magnus Gustafsson ; Marie Stå ; hlberg ; Gunvor Alvelius ; Bolette Husman ; Mats Carlquist ; Hans Jö ; rnvall ; and Tomas Bergman
- 刊名：Analytical Chemistry
- 出版年：2004
- 出版时间：October 1, 2004
- 年：2004
- 卷：76
- 期：19
- 页码：5864 - 5871
- 全文大小：171K
- 年卷期：v.76,no.19(October 1, 2004)
- ISSN：1520-6882
文摘
A compact disk (CD)-based microfluidic method forselective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinitychromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin andprocessed on the CD using nanoliter scale IMAC with andwithout subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allowsselective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da ormultiples of 80 Da). The CD contains 96 microstructures,each with a 16 nL IMAC microfluidic column. Movementof liquid is controlled by differential spinning of the disk.Up to 48 samples are distributed onto the CD in two equalsets. One set is for phosphopeptide enrichment only, theother for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are elutedfrom the columns directly into MALDI target areas, stillon the CD, using a solvent containing the MALDI matrix.After crystallization, the CD is inserted into a MALDI massspectrometer for analysis down to the femtomole level.The average success rate in phosphopeptide detection isover 90%. Applied to noncharacterized samples, themethod identified two novel phosphorylation sites, Thr735 and Ser 737, in the ligand-binding domain of thehuman mineralocorticoid receptor.