Phosphopeptide enrichment on functionalized polymer microspots for MALDI-MS analysis

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.

Lab‐on‐a‐plate: Extending the functionality of MALDI‐MS and LDI‐MS targets

“…Dunn, Reid, and Bruening (2010) enumerated several onplate protocols for phosphopeptides; immobilized metal affinity chromatography (IMAC) MALDI plates include: commercial affinity chips (e.g., the so-called ''ProteinChip'' commercialized by Ciphergen Biosystems, Fremont, CA), monolayers of immobilized metal-ion complexes, polymer-modified plates and zirconium phosphonate films; while MALDI plates modified with metal oxides include targets containing zirconium oxide and titanium dioxide. An exciting example of a method for the onplate enrichment of phosphopeptides is the one presented by Wang and Bruening (2009): the authors reported modification of silicon wafers with 250 mm-diameter microspots, which contain phosphopeptide-binding polymer brushes (Fig. 5).…”

“…The largest number of applications of the affinity-based onplate preconcentration of analytes is seen in the field of phosphoproteomics (e.g., Dunn et al, 2008;Blacken et al, 2009;Torta et al, 2009;Wang & Bruening, 2009). Dunn, Reid, and Bruening (2010) enumerated several onplate protocols for phosphopeptides; immobilized metal affinity chromatography (IMAC) MALDI plates include: commercial affinity chips (e.g., the so-called ''ProteinChip'' commercialized by Ciphergen Biosystems, Fremont, CA), monolayers of immobilized metal-ion complexes, polymer-modified plates and zirconium phosphonate films; while MALDI plates modified with metal oxides include targets containing zirconium oxide and titanium dioxide.…”

Phosphoproteomics: Searching for a needle in a haystack

“…A very low detection limit, a significantly improved phospho-profiling capability, and a simple experimental approach were reported. Wang and Bruening [43] described an interesting method for modification of Silicon wafers, which serve as MALDI plates with 250 μm-diameter microspots of phosphopeptide-binding polymer brushes enclosed by a hydrophobic poly(dimethylsiloxane) layer. Enrichment resulted in a 5-fold decrease in MALDI-MS detection limits and femtomole-level sensitivity.…”

Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

“…In order to enrich the phosphopeptides most of the new methods make use of the high affinity of the phosphoryl group to metals and metal oxides (e.g., on immobilized metal affinity chromatography (IMAC) [2][3][4][5] , metal oxide affinity chromatography (MOAC) 1,[6][7][8][9][10][11][12][13][14] , sequential immobilized metal affinity chromatography (SIMAC) 15 and direct on-target enrichment [16][17][18][19][20][21][22][23] ). Mass spectrometry (MS) is then commonly used to analyze the peptides.…”