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.

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

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.

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

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

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.

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:

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.

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.

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