Advances in Higher Order Multiplexing Techniques in Proteomics

J Proteome Res. 2019 Jun 7;18(6):2360-2369. doi: 10.1021/acs.jproteome.9b00228. Epub 2019 May 22.


Proteomics by mass spectrometry (MS) allows the large-scale identification and quantitation of the cellular proteins in a given biological context. Systems biology studies from proteomics data are largely limited by the accuracy and coverage of quantitative proteomics along with missing values. Toward this end, statistically robust biological observations are required, comprising multiple replicates, preferably with little technical variations. Multiplexed labeling techniques in proteomics allow quantitative comparisons of several biological samples or conditions. In this focused Review, we discuss an emerging technique called higher order multiplexing or enhanced multiplexing, a unique combination of traditional MS1- and MS2-based quantitative proteomics methods that allows for expanding the multiplexing capability of MS methods to save valuable instrument time, achieve statistical robustness, enhance coverage and quantitation accuracy, and reduce the run-to-run variability. We discuss the various innovative studies and experimental designs that exploit the power of this technique and its variants to provide an overview of a rapidly growing area and also to highlight the advantages and challenges that lie ahead in the widespread adoption of this technique.

Keywords: SILAC; TMT; cPILOT; hyperplexing; iTRAQ; multiplexing; proteome dynamics; quantitative proteomics.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Isotope Labeling
  • Mass Spectrometry
  • Proteome / genetics*
  • Proteomics*
  • Tandem Mass Spectrometry


  • Proteome