title: An improved sample preparation method for the sensitive detection of peptides by MALDI-MS.
abstract: We describe here an optimization study of the sample preparation conditions for sensitive detection of peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Among many factors in the conditions, we varied the percent acetonitrile in the peptide solution, the percent acetonitrile in the matrix solution and the α-cyano-4-hydroxycinnamic acid (CHCA) concentration in the matrix solution. CHCA was chosen because it is the most frequently used matrix for analyzing peptides. The well-established dried-droplet method was employed for sample deposition. The examined range of the concentration of CHCA was from 0.01 to 10 mg/ml, and the MeCN content of the solvent for matrix/analyte was 10% to 50%. The indicator for the detection sensitivity was the S/N ratio of the peaks of peptides used. Highly increased sensitivity (100- to 1000-fold) was observed for the optimal CHCA concentration of 0.1 mg/ml in 20% MeCN/0.1% aq. trifluoroacetic acid (TFA), as compared with the conventional concentration (10 mg/ml) in 50% MeCN/0.1% aq. TFA. For example, the limit of detection of human ACTH 18-39 was 10 amol/well for the optimal condition but 10 fmol/well for the conventional condition. The optimal condition (0.1 mg/ml CHCA in 20% MeCN/0.1% aq. TFA) was verified with five model peptides and provided significant improvement in sensitivity (by two to three orders of magnitude) compared with the conventional conditions. Optimizing the CHCA concentration and solvent composition significantly improved the detection sensitivity in the analysis of peptides by MALDI-MS.
authors.affiliation: Koichi Tanaka Laboratory of Advanced Science and Technology (KTLAST), Shimadzu Corporation, Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto, 604-8511, Japan.
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Hioki
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kuyama
authors.forename: Chikako
authors.initials: C
authors.lastname: Hamana
authors.forename: Kohei
authors.initials: K
authors.lastname: Takeyama
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000818:Animals; D002417:Cattle; D003373:Coumaric Acids / Q000737:chemistry; D057230:Limit of Detection; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000097:Acetonitriles; D003373:Coumaric Acids; D010455:Peptides; D012710:Serum Albumin, Bovine; C007175:alpha-cyano-4-hydroxycinnamate; C032159:acetonitrile
keywords: MALDI mass spectrometry; acetonitrile content; matrix concentration; peptide analysis; sensitivity