Development of a titanium dioxide nanoparticle pipette-tip for the selective enrichment of phosphorylated peptides.
The selective enrichment of specific proteins or peptides on micropipette tips prior to mass spectrometry analysis, which can minimize non-specific interferences as well as sample loss, has been an important issue in current proteomics field. In this paper, we have developed an easy-to-use phosphopeptide-selective pipette tip in which titanium dioxide nanoparticles were embedded in monolithic structure photopolymerized from ethylene glycol dimethacrylate. The simple and convenient fabrication was feasible in a commercial polypropylene pipette tip. Phosphorylated peptides were isolated from non-phosphopeptides by TiO(2) nanoparticle and eluted by 100 mM ammonium phosphate (pH 8.5), which was compatible with 2,5-dihydroxybenzoic acid (DHB)/1% phosphoric acid matrix and allowed for direct analysis of the elution fraction by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) without the necessity of desalting pretreatment. Tryptic digested alpha-casein and beta-casein spiked into bovine serum albumin (BSA) nonphosphorylated peptides (molar ratio 1:1:10) were used to assess the selectivity of TiO(2) tips. The effect of 50 mM ammonium hydrogencarbonate, pH 8 in 50% acetonitrile used as a wash buffer in reduction of nonspecific bound peptide to TiO(2) tip was dramatic. Almost all non-phosphopeptides were not detected by MALDI-MS analysis. The lowest detectable amount of phosphopeptide was estimated at low femtomole level. The easy-to-use TiO(2)-embeded tips operated in combination with the modified wash and elution conditions enable an efficient phosphopeptide enrichment for mass spectrometric analysis.
Hsieh
Hui-Ching
HC
Life Science Business Unit, C SUN MFG. LTD., 7F-9, No. 79, Hsin Tai Wu Rd., Sec. 1, Hsi-Chin, Taipei Hsien, Taiwan.
Sheu
Calvin
C
Shi
Fong-Ku
FK
Li
Ding-Tzai
DT
D002364:Caseins; D002846:Chromatography, Affinity / Q000379:methods*; D053758:Nanoparticles / Q000648:ultrastructure; D010748:Phosphopeptides / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014025:Titanium
D002364:Caseins; D010748:Phosphopeptides; C009495:titanium dioxide; D012710:Serum Albumin, Bovine; D014025:Titanium