Enhanced Multiplexing Technology for Proteomics

Abstract

The identification of thousands of proteins and their relative levels of expression has furthered understanding of biological processes and disease and stimulated new systems biology hypotheses. Quantitative proteomics workflows that rely on analytical assays such as mass spectrometry have facilitated high-throughput measurements of proteins partially due to multiplexing. Multiplexing allows proteome differences across multiple samples to be measured simultaneously, resulting in more accurate quantitation, increased statistical robustness, reduced analysis times, and lower experimental costs. The number of samples that can be multiplexed has evolved from as few as two to more than 50, with studies involving more than 10 samples being denoted as enhanced multiplexing or hyperplexing. In this review, we give an update on emerging multiplexing proteomics techniques and highlight advantages and limitations for enhanced multiplexing strategies. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

A Tutorial Review of Labeling Methods in Mass Spectrometry-Based Quantitative Proteomics

“…94 Other methods have also been reported recently with different tag combinations. 95,96 For further insights into these methods, we recommend reading the review article by Bowser et al 97 One significant advantage of hybrid labeling methods is the substantial increase in throughput. This approach can save considerable instrument time and reduce sample preparation variability.…”

Evaluating cPILOT Data toward Quality Control Implementation

“…Enhanced multiplexing technologies such as N -hydroxysuccinimide-ester tandem labeling in one pot (NETLOP), 29-plex tandem mass tag (TMT), TAG-TMTpro, combined precursor isotopic labeling and isobaric tagging (cPILOT), and others − enable high-throughput proteomics experiments by combining more than one multiplexing technology into a single experiment. For example, NETLOP combines isotopic and isobaric versions of TMT tags to achieve up to 48-plex labeling, TAG-TMTpro combines amino acid labeling with TMT isobaric tags to achieve up to 45-plex labeling, and cPILOT combines isotopic dimethylation and TMT or dimethyl leucine (DiLeu) isobaric tags to achieve up to 42-plex labeling. − ,,, cPILOT can encompass multiple conditions such as tissue types, time points, and genotypes and has been broadly applied to study aging, Alzheimer’s disease, ,, and post-translational modifications . cPILOT is attractive due to its ability to increase quantitative accuracy and reduce data acquisition times, experimental costs, and sample error.…”

“…Quantitative proteomics aims to measure relative and absolute proteome changes across various experimental conditions to expand understanding of disease, mechanistic pathways, and biological processes. 1 Enhanced multiplexing technologies such as N-hydroxysuccinimide-ester tandem labeling in one pot (NETLOP), 2 29-plex tandem mass tag (TMT), 3 TAG-TMTpro, 4 combined precursor isotopic labeling and isobaric tagging (cPILOT), 5 and others 6−17 enable high-throughput proteomics experiments by combining more than one multiplexing technology into a single experiment. For example, NETLOP combines isotopic and isobaric versions of TMT tags to achieve up to 48-plex labeling, TAG-TMTpro combines amino acid labeling with TMT isobaric tags to achieve up to 45-plex labeling, and cPILOT combines isotopic dimethylation and TMT or dimethyl leucine (DiLeu) isobaric tags to achieve up to 42-plex labeling.…”

“…For example, NETLOP combines isotopic and isobaric versions of TMT tags to achieve up to 48-plex labeling, TAG-TMTpro combines amino acid labeling with TMT isobaric tags to achieve up to 45-plex labeling, and cPILOT combines isotopic dimethylation and TMT or dimethyl leucine (DiLeu) isobaric tags to achieve up to 42-plex labeling. [1][2][3][4]6,7,18 cPILOT can encompass multiple conditions such as tissue types, time points, and genotypes and has been broadly applied to study aging, 19 Alzheimer's disease, 18,20,21 and post-translational modifications. 22 cPILOT is attractive due to its ability to increase quantitative accuracy and reduce data acquisition times, experimental costs, and sample error.…”

“…Quantitative proteomics aims to measure relative and absolute proteome changes across various experimental conditions to expand understanding of disease, mechanistic pathways, and biological processes . Enhanced multiplexing technologies such as N -hydroxysuccinimide-ester tandem labeling in one pot (NETLOP), 29-plex tandem mass tag (TMT), TAG-TMTpro, combined precursor isotopic labeling and isobaric tagging (cPILOT), and others − enable high-throughput proteomics experiments by combining more than one multiplexing technology into a single experiment.…”