Photopolymerized microtips for sample preparation in proteomic analysis

Abstract

We demonstrate a novel method for the fabrication of disposable plastic microtips, which we name "EasyTip", by a photopolymerization technique. C18 reversed-phase (C18) and ion metal affinity chromatography (IMAC) beads were immobilized on a plastic pipette tip, made of polypropylene materials, by photo-initiated polymerization. The fabricated EasyTips can be manipulated using commercial pipettes for wash/elution of minute amount of biological samples (< 10 microL) and can be applied for mass spectrometry (MS)-based proteomic analysis, in which the detection sensitivity depends critically on the optimal sample preparation. The recovery of a sample of 25 fmol of tryptic hemoglobin digest loaded in a C18 EasyTip was near 100% and we estimated the loading capacity to be around 0.4-2.0 microg of total proteins or peptides, which is well above a sufficient quantity for MS analysis. The effectiveness of the C18 EasyTips in enhancing the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MS signal, and thus providing a greater sequence coverage, was also demonstrated by the analysis of hemoglobin digest and the in-gel digested epidermal growth factor receptor (EGFR) protein from A431 cell lysate. We also demonstrated the usefulness of the immobilized IMAC EasyTips in extracting the signal of tryptic phosphopeptides of beta-casein (10 pmol) having one and four phosphorylation sites by using an IMAC EasyTip prior to off-line analysis by MS. The combination of IMAC EasyTips and MALDI-MS allowed the unambiguous identification of phosphopeptides based on the phosphatase assay as well as the post-source decay. Compared to other miniaturized devices, this fabrication method is simple, cheap, and requires less human intervention. Moreover, the method of manipulating the EasyTips is straightforward and can be automated readily by a robotic system for high-throughput analysis.