Scrambled Internal Standard Method for High-Throughput Protein Quantification by Matrix-Assisted Laser Desorption Ionization Tandem Mass Spectrometry.
Matrix-assisted laser desorption ionization (MALDI) could be advantageous for high-throughput MS acquisition but suffers from low signal reproducibility. The purpose of this study was to establish a reliable MALDI-tandem mass spectrometry (MS/MS)-based high-throughput quantification of tryptic peptides using our newly developed scrambled internal standard (sIS) method. The standard curves obtained with sIS peptides showed good linearity over a wide concentration range (5-1000 fmol/μL) compared to that with the IS-free method, and the coefficient of variation of data points at each concentration (5-1000 fmol/μL) was significantly reduced. Furthermore, the ion suppression effect of digested serum could be normalized with the sIS peptides. Differences of quantitative values obtained by MALDI-MS/MS and liquid chromatography-MS/MS with selected reaction monitoring were within 20% in the presence of 0.1-5 μL of immunoprecipitated model plasma. Furthermore, the effect of amino acid composition on peptide sensitivity was examined, and we found that sensitivity was significantly decreased if an aromatic amino acid was replaced with a nonaromatic amino acid. Thus, high sensitivity required the use of sIS peptides containing an aromatic amino acid. Finally, the sIS method enabled high-throughput quantification of tryptic peptides with high accuracy and a wide dynamic range.
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Toshihiro
T
Yoneyama
Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University , 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.|Japan Agency for Medical Research and Development (AMED) CREST , Tokyo 100-0004, Japan.
Sumio
0000-0003-4634-7133
S
Ohtsuki
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Masanori
M
Tachikawa
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Yasuo
Y
Uchida
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Tetsuya
T
Terasaki
D000595:Amino Acid Sequence / Q000235:genetics; D002853:Chromatography, Liquid; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010455:Peptides; D011506:Proteins
MALDI; MS/MS; absolute quantification; high-throughput; plasma; scrambled internal standard