Protein Preparation and Separation

Protein extraction, modification and separation protocols

Protein extraction is an important step in any proteomics experiment. It often starts with cell lysis and cell fractionation, followed by specific enrichment and/or isolation of a particular protein of interest (affinity purification), or removal of interfering or contaminating substances (i.e. immunodepletion).

Many techniques are available for the disruption of cells, including physical and detergent-based methods. Historically, physical lysis has been the method of choice for cell disruption; however, physical methods often require expensive equipment (french prss, homogenizer, cryogrinding).
Detergent-based lysis is more popular due to ease of use, low cost and efficient protocols. However, many detergents interfere with the downstream LC-MS analysis. A list of MS compatible detergents can be found on our protein digestion page.

Sample Preparation Overviews

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Workflow for Protein Mass Spectrometry from Thermopage
Protein Sample Preparation for Mass Spectrometry page
Mass Spectrometry Sample Preparation Handbook page
Protein Purification and Isolation Support Center page
Physical cell lysis page
Detergent based cell lysis page
Sigma's Proteomics page
Tutorials from the Broad’s Proteomics Platform page

Cell culture related protocols

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Protein Extraction from Tissues and Plants

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Protein preparation from Serum, Plasma and Biofluids

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Isotopic labelling strategies

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SILAC (stable isotope labeling using amino acids in cell culture) is a metabolic labelling technique for comprehensive identification, characterization and quantification of proteins by LC-MS/MS. Isotopically labeled amino acids (typically Lys and Arg) are incorporated in to proteins during cell culture.
There are various chemical labeling strategies for concurrent peptide identification and multiplexed proteomics quantitation by mass spectrometry. Most quantitative proteomics reagents incorporate stable isotopes into the isobaric tag portion of the reagents and are used to label at the protein or peptide level:

Crosslinking strategies

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Protein Separation and Enrichment

The most challenging proteomics studies are those that try to identify and quantify global proteomes. Many experiments require a reduction of protein complexity by separating the proteins.
One approach is to focus on the subproteome of an organelle using classical subcellular fractionation techniques.
Alternatively, sample complexity can also be effectively reduced using enrichment techniques like immunoprecipitations or activity- or affinity-based approaches.
Enrichment can also be directed toward a specific characteristic of proteins for example toward glycoproteins [Ref 2], phoshoproteins or newly synthesized proteins [Ref 3].
Proteins can also be separated by gels (SDS-PAGE), chromatography (FPLC and HPLC) or Gel-Free separations.

Affinity-activity based protein enrichment

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Proteins can be enriched based on a specific interactions. Either the bait or the protein of interest are immobilized to extract the proteins of interest. All those methods have to be carefully optimized in terms of bait and sample load with appropriate positive and negative controls.

Immunoprecipitation protocols

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Immunoprecipitation (IP) is the most commonly used affinity purification of antigen using a specific antibody. Overview of the Immunoprecipitation (IP) Technique

Tips:
  • To reduce antibody contamination in your final sample consider following a protocol which immobilizes the antibody (via crosslinking)
  • Avoid using detergents as much as possible.
  • Use an elution buffer compatible with the downstream analysis. For mass spec analysis use Urea buffer (4-6 M, pH 7.5) to be compatible with reduction/alkylation and tryptic digestion, and desalt prior to LC-MS analysis.

    Note: choose a mass spec compatible elution buffer to allow for in solution digestion and minimal peptide cleanup

Tag based affinity protocols

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Affinity tag and tandem affinity tag (TAP) allow to isolate specific proteins of interest and proteins associated with them. Commonly used affinity tags used in combination with an antibody towards either of these tags include:
FLAG; peptide sequence DYKDDDDK
c-Myc; peptide sequence EQKLISEEDL
Hemagglutinin (HA); peptide sequence YPYDVPDYA
V5; peptide sequence GKPIPNPLLGLDST
Green fluorescent protein (GFP)
Poly His tag
GST tag
Proteins tagged with biotin can be enriched using streptavidin or neutravidin beads.

Immunodepletion

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The wide dynamic range of protein concentrations present in serum and plasma represents a major challenge for the analysis of the proteome. The range of plasma protein concentrations, from low abundance proteins such as cytokines (pg/mL) to albumin (30-45 mg/mL), represents at least 10 orders of magnitude. Analysis of low abundance proteins by even the most sensitive of methods for proteomics greatly benefits from the removal of high abundance proteins that may interfere with their detection. Plasma Immunodepletion strategies specifically remove the most abundant proteins from human plasma or serum in preparation for further proteomics analysis, enabling deeper penetration into the plasma proteome, here are a couple of examples:

Cell Fractionation (organellar proteomics)

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When fractionating proteins based on their cellular localization all possible precautions should be taken to isolate the desired organelle. Contaminating proteins from other cytosol or other organelles should be expected. Repeated analyses with strong statistical tests are often needed to differentiate contaminating proteins from true organellar proteins. Another (undesired) organelle can serve as a good negative control.

Protein separation 1D and 2D Gel

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a very common method for separating proteins based on their molecular weight. Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative abundance of major proteins in a sample, and to determine the distribution of proteins among fractions. SDS-PAGE can also be used as a tool to assess purity of protein samples and to monitor the progress of a fractionation or purification procedure. Two-dimensional (2D) electrophoresis separated proteins first by their isoelectric point and then by their molecular weight.
Different staining methods can be used like Coomassie Blue ( range) and Silver stain (range) to visualize the protein bands. Techniques such as Western blotting are used to detect specific proteins or protein modification.
To indetify proteins by LC-MS, proteins can be digested in the gel or on the membrane (western blot) and peptides can be extracted for analysis.

Chromatography HPLC or FPLC

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Traditional column chromatography or using an HPLC (High-performance liquid chromatography) or FPLC (Fast protein liquid chromatography) can be used to separate proteins based on their physiochemical properties like size, charge and hydrophobicity; Size exclusion, cation or anion exchange, reversed phase (C4) are some of the commonly used chemistries.

Gel-Free (Free-Flow-Electrophoresis
less common and a bit tricky.. BD recently made the decision to exit the Free Flow Electrophoresis (FFE) business, but Expedeon is still offering a Gel Free System

References

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  1. Ligand-based receptor identification on living cells and tissues using TRICEPS. Frei AP, Moest H, Novy K, Wollscheid B. Nat Protoc. 2013 Jul;8(7):1321-36. doi: 10.1038/nprot.2013.072. Epub 2013 Jun 13. link
  2. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Zhang H, Li XJ, Martin DB, Aebersold R.Nat Biotechnol. 2003 Jun;21(6):660-6. link
  3. Dopaminergic modulation of the hippocampal neuropil proteome identified by bio-orthogonal non-canonical amino-acid tagging (BONCAT). J.J. Hodas et al., Proteomics 12, 2464-2476 (2012). link
  4. Proteomics of human plasma: A critical comparison of analytical workflows in terms of effort, throughput and outcome. Loȉc Dayon, Martin Kussmann EuPA Open Proteomics Volume 1, 2013, Pages 8-16 12, 2464-2476 (2012). link