title: Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis.
abstract: Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.
authors.affiliation: Department of Applied Chemistry, National Chi Nan University, Puli, Nantou 545, Taiwan.
authors.forename: Kai Jung
authors.initials: KJ
authors.lastname: Shiau
authors.forename: Shain-Un
authors.initials: SU
authors.lastname: Hung
authors.forename: Hsiao-Wen
authors.initials: HW
authors.lastname: Lee
authors.forename: Chih-Che
authors.initials: CC
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000302:isolation & purification; D002417:Cattle; D006801:Humans; D008969:Molecular Sequence Data; D058612:Nanodiamonds / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014025:Titanium / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D002364:Caseins; D058612:Nanodiamonds; D010455:Peptides; D010748:Phosphopeptides; C009495:titanium dioxide; C015462:polyarginine; D012710:Serum Albumin, Bovine; D014025:Titanium; D014357:Trypsin