A Standardized and Reproducible Proteomics Protocol for Bottom-Up Quantitative Analysis of Protein Samples Using SP3 and Mass Spectrometry

Methods Mol Biol. 2019:1959:65-87. doi: 10.1007/978-1-4939-9164-8_5.


The broad utility of mass spectrometry (MS) for investigating the proteomes of a diverse array of sample types has significantly expanded the use of this technology in biological studies. This widespread use has resulted in a substantial collection of protocols and acquisition approaches designed to obtain the highest-quality data for each experiment. As a result, distilling this information to develop a standard operating protocol for essential workflows, such as bottom-up quantitative shotgun whole proteome analysis, can be complex for users new to MS technology. Further complicating this matter, in-depth description of the methodological choices is seldom given in the literature. In this work, we describe a workflow for quantitative whole proteome analysis that is suitable for biomarker discovery, giving detailed consideration to important stages, including (1) cell lysis and protein cleanup using SP3 paramagnetic beads, (2) quantitative labeling, (3) offline peptide fractionation, (4) MS analysis, and (5) data analysis and interpretation. Special attention is paid to providing comprehensive details for all stages of this proteomics workflow to enhance transferability to external labs. The standardized protocol described here will provide a simplified resource to the proteomics community toward efficient adaptation of MS technology in proteomics studies.

Keywords: Bottom-up; Mass spectrometry; Paramagnetic beads; Protein cleanup; Protein digestion; Proteomics; Quantification; SP3; Tandem mass tagging.

MeSH terms

  • Chromatography, High Pressure Liquid
  • Chromatography, Liquid
  • Data Interpretation, Statistical
  • Humans
  • Magnetite Nanoparticles*
  • Mass Spectrometry* / methods
  • Peptides
  • Proteome
  • Proteomics* / methods
  • Proteomics* / standards
  • Workflow


  • Magnetite Nanoparticles
  • Peptides
  • Proteome