Facile Preparation of Peptides for Mass Spectrometry Analysis in Bottom-Up Proteomics Workflows

Melinda Wojtkiewicz, Linda Berg Luecke, Maia I. Kelly, Rebekah L. Gundry

Research output: Contribution to journalArticlepeer-review

Abstract

Mass spectrometry (MS) is routinely used to identify, characterize, and quantify biological molecules. For protein analysis, MS-based workflows can be broadly categorized as top-down or bottom-up, depending on whether the proteins are analyzed as intact molecules or first digested into peptides. This article outlines steps for preparing peptide samples for MS as part of a bottom-up proteomics workflow, providing versatile methods suitable for discovery and targeted analyses in qualitative and quantitative workflows. Resulting samples contain peptides of suitable size for analysis by MS instrumentation generally available to modern research laboratories, including MS coupled to either liquid chromatography (LC) or matrix-assisted laser desorption/ionization (MALDI) interfaces. This article incorporates recent developments in methodologies and consumables to facilitate sample preparation. The protocols are well-suited to users without prior experience in proteomics and include methods for universally applicable suspension trap processing and for alternate in-solution processing to accommodate a range of sample types. Cleanup, quantification, and fractionation procedures are also described.

Original languageEnglish (US)
Article numbere85
JournalCurrent Protocols
Volume1
Issue number3
DOIs
StatePublished - Mar 2021

Keywords

  • bottom-up proteomics
  • detergent removal
  • mass spectrometry
  • S-Trap™

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Health Informatics
  • Medical Laboratory Technology

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