TY - JOUR
T1 - Scrambled Internal Standard Method for High-Throughput Protein Quantification by Matrix-Assisted Laser Desorption Ionization Tandem Mass Spectrometry
AU - Yoneyama, Toshihiro
AU - Ohtsuki, Sumio
AU - Tachikawa, Masanori
AU - Uchida, Yasuo
AU - Terasaki, Tetsuya
N1 - Funding Information:
This study was supported in part by the Industrial Technology Research Grant Program from the New Energy and the Industrial Technology Development Organization of Japan and a research program in the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct) Ministry of Education, Culture Sports, Science and Technology of Japan. This study was also supported in part by JSPS KAKENHI Grant 14J05219 and an Applied Research for Innovative Treatment of Cancer grant by the Ministry of Health, Labour and Welfare of Japan, and AMED-CREST by the Japan Agency for Medical Research and Development. Data was collected by the MALDI-linear ion trap quadrupole (LTQ XL) mass spectrometer from Thermo Fisher Scientific. Providers of funding and Thermo Fisher Scientific had no role in the design of the study, collection of the data, analysis or interpretation of the data, decision to submit the manuscript for publication, or writing of the manuscript.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/4/7
Y1 - 2017/4/7
N2 - 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.
AB - 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.
KW - MALDI
KW - MS/MS
KW - absolute quantification
KW - high-throughput
KW - plasma
KW - scrambled internal standard
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U2 - 10.1021/acs.jproteome.6b00941
DO - 10.1021/acs.jproteome.6b00941
M3 - Article
C2 - 28317378
AN - SCOPUS:85017146181
VL - 16
SP - 1556
EP - 1565
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 4
ER -