期刊
ANALYST
卷 134, 期 3, 页码 512-518出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/b815598d
关键词
-
资金
- National Science Foundation [CHE0616795]
- Michigan State University [HBRI-639]
On-plate enrichment of phosphopeptide digests followed by MALDI-MS is attractive for analyzing small quantities of phosphoproteins because it involves minimal sample handling and reduces sample loss. This work describes a method for modification of Si wafers, which serve as MALDI plates, with 250 mm-diameter microspots of phosphopeptide-binding polymer brushes enclosed by a hydrophobic poly(dimethylsiloxane) (PDMS) layer. Formation of the patterned surface occurs by heating a patterned PDMS stamp on a Si wafer, growing the polymer brushes from the Si exposed in the stamp pores, and removing the stamp to leave behind a residual layer of PDMS surrounding the brushes. Pinning and evaporation of a sample droplet on the microspot concentrate samples and enrich phosphopeptides through binding to the small area. After rinsing with acidic solution to remove unwanted peptides, a drop of matrix solution elutes the phosphopeptides and crystallizes only on the microspot. With beta-casein and ovalbumin digests, the use of microspots rather than 2 mm-diameter polymer spots for phosphopeptide enrichment results in a 5-fold decrease in MALDI-MS detection limits and low-femtomole level sensitivity. Improved enrichment of phosphopeptides on the polymer microspots from samples that contain a 10-fold molar excess of peptides from a non-phosphorylated protein digest is also possible with the help of a sonication-assisted rinse. Thus, arrays of microspots are attractive for analyzing femtomole amounts of relatively pure protein, such as that obtained by immunoprecipitation.
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