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Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides
Published on: July 14, 2015
Phosphopeptide enrichment on functionalized polymer microspots for MALDI-MS analysis
Wei-Han Wang1, Merlin L Bruening
1Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Abstract:
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 microm-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.
Insights
This study presents a novel method for phosphopeptide enrichment using polymer brush microspots on MALDI plates, achieving low-femtomole sensitivity for phosphoprotein analysis.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Materials Science
Background:
- On-plate enrichment of phosphopeptides using MALDI-MS offers advantages in analyzing limited protein quantities.
- Current methods can be limited by sample handling and loss.
- Developing sensitive and efficient enrichment techniques is crucial for phosphoproteomics.
Purpose of the Study:
- To develop a method for on-plate phosphopeptide enrichment using patterned polymer brushes on MALDI plates.
- To improve sensitivity and reduce detection limits for phosphopeptide analysis.
- To enable the analysis of femtomole amounts of phosphoproteins.
Main Methods:
- Modification of silicon (Si) wafers with 250-micrometer diameter microspots of phosphopeptide-binding polymer brushes.
- Patterning using a hydrophobic poly(dimethylsiloxane) (PDMS) stamp.
- Utilizing microspot geometry for sample concentration and phosphopeptide binding.
- Employing acidic rinses and matrix solution for elution and MALDI-MS analysis.
Main Results:
- Achieved a 5-fold decrease in MALDI-MS detection limits compared to larger spots.
- Demonstrated low-femtomole level sensitivity for phosphopeptide enrichment.
- Showed improved enrichment of phosphopeptides from complex mixtures with a 10-fold molar excess of non-phosphorylated peptides.
- Sonication-assisted rinsing enhanced enrichment efficiency.
Conclusions:
- Patterned polymer brush microspots on Si wafers are effective for on-plate phosphopeptide enrichment.
- This method significantly enhances sensitivity and reduces detection limits in MALDI-MS analysis.
- Arrays of microspots are suitable for analyzing femtomole quantities of relatively pure proteins, particularly those obtained via immunoprecipitation.
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