Tmt sample preparation for proteomics facility submission and subsequent data analysis

Silveli Suzuki-Hatano, Ang Chen Tsai, Audrey Daugherty, Christina A. Pacak

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

Original languageEnglish (US)
Article numbere60970
Pages (from-to)1-6
Number of pages6
JournalJournal of Visualized Experiments
Volume2020
Issue number160
DOIs
StatePublished - 2020
Externally publishedYes

Bibliographical note

Funding Information:
We would like to acknowledge the UF-ICBR proteomics facility for their processing of our samples. This work was supported in part by the National Institutes of Health R01 HL136759-01A1 (CAP).

Publisher Copyright:
© 2020, Journal of Visualized Experiments. All rights reserved.

Keywords

  • Biochemistry
  • Issue 160
  • Multiplex proteomics
  • Processing proteomics data
  • Protein dysregulation
  • Protein sample preparation
  • Quantitative proteomics
  • Tandem mass tag
  • Untargeted proteomics

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Video-Audio Media

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