High-throughput protein characterization using mass spectrometric immunoassay

Urban A. Kiernan, Kemmons A. Tubbs, Karl Gruber, Dobrin Nedelkov, Eric E. Niederkofler, Peter Williams, Randall W. Nelson

Research output: Contribution to journalArticle

70 Scopus citations

Abstract

A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity pipette tips for subsequent use in the extraction of specific proteins from plasma and deposition onto 96-well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) targets. Samples from multiple individuals were screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma samples for the transthyretin-associated transport protein, retinol-binding protein (RBP). Analyses were able to detect the presence of posttranslationally modified TTR and RBP, as well as a mutation present in the TTR of one individual. Subsequent analyses of wild-type and mutated TTR using enzymatically active MALDI-TOF MS targets were able to identify the site and nature of the point mutation. The approach represents a rapid (∼100 samples/2 h, reagent preparation -to-data) and accurate means of characterizing specific proteins present in large numbers of individuals for proteomic and clinical/diagnostic purposes.

Original languageEnglish (US)
Pages (from-to)49-56
Number of pages8
JournalAnalytical Biochemistry
Volume301
Issue number1
DOIs
StatePublished - Feb 1 2002

Keywords

  • Affinity
  • High-throughput
  • Immunoassay
  • MALDI-TOF
  • Mass spectrometry
  • Protein

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Kiernan, U. A., Tubbs, K. A., Gruber, K., Nedelkov, D., Niederkofler, E. E., Williams, P., & Nelson, R. W. (2002). High-throughput protein characterization using mass spectrometric immunoassay. Analytical Biochemistry, 301(1), 49-56. https://doi.org/10.1006/abio.2001.5478