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High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

Aleksandr Gaun1, Niclas Olsson1, John C K Wang1

  • 1Calico Life Sciences LLC, South San Francisco, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|February 13, 2023
PubMed
Summary

We developed a high-throughput method for co-fractionation mass spectrometry (CF-MS) to profile native plasma protein complexes. This technique enables detailed analysis of protein interactions in various biological states for comprehensive proteome profiling.

Keywords:
Isobaric labelingNative separationPlasmaProtein complexomeProtein-protein interactionsTMT/tandem mass tag

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Area of Science:

  • Biochemistry
  • Proteomics
  • Analytical Chemistry

Background:

  • Proteins form dynamic macromolecular complexes crucial for cellular functions.
  • Alterations in protein complexes are associated with various disease and cell states.
  • Understanding native protein interactions is vital for biological and medical research.

Purpose of the Study:

  • To present a high-throughput method for co-fractionation mass spectrometry (CF-MS) for native plasma protein profiling.
  • To detail sample preparation for analyzing endogenous protein complexes.
  • To offer strategies for comprehensive proteome profiling.

Main Methods:

  • High-throughput sample preparation for co-fractionation mass spectrometry (CF-MS).
  • Separation of 18 plasma samples into 954 size exclusion chromatography (SEC) fractions.
  • Implementation of both label-free and isobaric labeling (TMTpro) approaches.

Main Results:

  • Successful generation of 954 SEC fractions from 18 plasma samples.
  • Detailed protocols for automated sample preparation for high-throughput analysis.
  • Demonstration of adaptable methodology for varying sample numbers.

Conclusions:

  • The described CF-MS method provides a robust platform for high-throughput native plasma proteome profiling.
  • Combining label-free and isobaric labeling strategies enhances comprehensive analysis of protein complexes.
  • This approach facilitates the study of protein interactions in different biological contexts.