Digestion, Purification, and Enrichment of Protein Samples for Mass Spectrometry

Victoria E. Hedrick1, Mercedes N. LaLand1, Ernesto S. Nakayasu1, Lake N. Paul1

1 Bindley Bioscience Center, Purdue Proteomics Facility, Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140272
Online Posting Date:  September, 2015
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Proteomic studies rely heavily on the use of liquid chromatography (LC)–mass spectrometry (MS and MS/MS) analyses to provide information about protein composition and function. Profiling the proteome can be the first step to understanding biological pathways, but the challenges scientists face with the complex nature of proteins and proteolysis products can be daunting. Techniques involving fractionation, immunoprecipitation, and phosphopeptide enrichment can simplify complex protein mixtures and enhance the amount of target proteins that are important to the investigator. Emphasis on sample preparation for LC‐MS/MS analyses is essential to acquisition of high‐quality data for proteomic research. Certain classes of reagents, materials, and contaminants that can be introduced during sample processing may limit the effectiveness of LC‐MS/MS analysis. These protocols outline methods for proteolytic digestion of proteins that are compatible with LC‐MS/MS, along with procedures that allow for simplification of complex protein matrices. © 2015 by John Wiley & Sons, Inc.

Keywords: peptides; phosphopeptide enrichment; protein extraction; fractionation; on‐bead digestion; in‐gel digestion

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Table of Contents

  • Introduction
  • Basic Protocol 1: In‐Solution Digestion of a Protein Mixture
  • Alternate Protocol 1: In‐Gel Digestion of a Protein Mixture
  • Alternate Protocol 2: Immunoprecipitation and On‐Bead Digestion of Protein‐Interaction Complexes
  • Basic Protocol 2: Liquid‐Liquid Extraction of Proteins Using Acetone
  • Alternate Protocol 3: Solid‐Phase Extraction of Proteins
  • Basic Protocol 3: Peptide Fractionation for 2D LC‐MS/MS Analysis
  • Basic Protocol 4: Phosphopeptide Enrichment
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: In‐Solution Digestion of a Protein Mixture

  • Protein sample
  • Milli‐Q‐purified water
  • Acetone, HPLC grade, −20°C
  • Ammonium bicarbonate (ABC, Sigma‐Aldrich), pH ∼8.1
  • Dithiothreitol (DTT, Sigma‐Aldrich)
  • Urea (reagent grade, 98%; Sigma‐Aldrich, optional)
  • Acetonitrile (ACN), HPLC grade
  • Triethylphosphine (TEP, Sigma‐Aldrich)
  • Iodoethanol (IEtOH, Sigma‐Aldrich)
  • Lys‐C/trypsin mixture, sequencing grade (Promega)
  • Vacuum centrifuge
  • Axygen Maximum Recovery Microtubes
  • Barocycler (Pressure Biosciences)
CAUTION: Use proper protection (hood, safety glasses, gloves, lab coat) when handling iodoethanol and triethylphosphine. IEtOH is a toxic and combustible liquid. TEP is pyrophoric (highly flammable) as a liquid and vapor. Both compounds must be handled in accordance with the material safety data sheets (MSDS).

Alternate Protocol 1: In‐Gel Digestion of a Protein Mixture

  Additional Materials (also see protocol 1)
  • SDS polyacrylamide gel containing protein of interest
  • Potassium ferricyanide (K 3Fe(CN) 6), ACS grade (Sigma‐Aldrich)
  • Sodium sulfite (Na 2SO 3), reagent grade (Sigma‐Aldrich)
  • Iodoacetamide (IAA, Sigma‐Aldrich)
  • Trifluoroacetic acid (TFA, Sigma‐Aldrich)
  • Acetonitrile (ACN), HPLC grade
  • Formic acid (Fluka)
  • Razor blade or scalpel
  • 55°C shaking water bath or heat block
  • 0.5‐ml microcentrifuge tube (e.g., Axygen Maximum Recovery)

Alternate Protocol 2: Immunoprecipitation and On‐Bead Digestion of Protein‐Interaction Complexes

  Additional Materials (also see protocol 1)
  • Sample of bead‐bound Co‐IP complexes (antibody‐antigen‐interacting partners)
  • Trypsin, sequencing grade (Sigma‐Aldrich), optional

Basic Protocol 2: Liquid‐Liquid Extraction of Proteins Using Acetone

  • Chloroform (CHCl 3), HPLC grade (Sigma‐Aldrich)
  • Methanol (MeOH), HPLC grade
  • Digested peptide sample (see protocol 1 or protocol 2 or 2)
  • Acetone, HPLC grade (Sigma‐Aldrich), −20°C
  • Vacuum centrifuge

Alternate Protocol 3: Solid‐Phase Extraction of Proteins

  Additional Materials (also see protocol 4)
  • C18 column: silica‐based C18 UltraMicroSpin Column Kit (The Nest Group)
  • Acetonitrile (ACN), HPLC grade
  • Digested peptide sample (see protocol 1 or protocol 2 or 2)
  • Milli‐Q‐purified water
  • Formic acid (Fluka)

Basic Protocol 3: Peptide Fractionation for 2D LC‐MS/MS Analysis

  • Protein sample or digested peptides (see Basic Protocols protocol 11 protocol 42 or Alternate Protocols protocol 21 protocol 53)
  • Milli‐Q‐purified water
  • Mobile phase A: 10 mM ammonium formate (Sigma‐Aldrich), pH 10.0, in water
  • Mobile phase B: 10 mM ammonium formate, pH 10.0, in 90% acetonitrile (ACN, HPLC grade)
  • Formic acid (Fluka)
  • High‐performance liquid chromatography (HPLC) equipment with UV detector, fraction collector, and XBridge C18 column (250 × 4.6 mm, 5‐μm particles, Waters)
  • 96‐well plates
  • Vacuum centrifuge

Basic Protocol 4: Phosphopeptide Enrichment

  • Magnetic nickel‐nitriloacetic acid (Ni‐NTA) agarose beads, 5% suspension (Qiagen, cat. no. 36111)
  • Milli‐Q‐purified water
  • 100 mM EDTA, pH 8.0 (Sigma‐Aldrich)
  • Ferric chloride (FeCl 3, Sigma‐Aldrich)
  • Acetonitrile (ACN), HPLC grade
  • Methanol (MeOH), HPLC grade
  • Acetic acid, ACS grade (Sigma‐Aldrich)
  • Peptide sample
  • Trifluoroacetic acid (TFA, Sigma‐Aldrich)
  • Ammonium hydroxide (NH 4OH), ACS grade (20‐30%, Mallinckrodt)
  • Microcentrifuge tubes
  • Magnetic tube rack
  • Tube rotator rotator
  • pH test strips
  • Vacuum centrifuge
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Internet Resources
  Salt tolerance table from the Scripps Center for Metabolomics and Mass Spectrometry.
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