Scrambled Internal Standard Method for High-Throughput Protein Quantification by Matrix-Assisted Laser Desorption Ionization Tandem Mass Spectrometry

Toshihiro Yoneyama, Sumio Ohtsuki, Masanori Tachikawa, Yasuo Uchida, Tetsuya Terasaki

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)1556-1565
Number of pages10
JournalJournal of Proteome Research
Volume16
Issue number4
DOIs
Publication statusPublished - 2017 Apr 7

Keywords

  • MALDI
  • MS/MS
  • absolute quantification
  • high-throughput
  • plasma
  • scrambled internal standard

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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