Selected Reaction Monitoring

Description

Summary of Method

  1. We use an LC-tandem mass spectrometry technique called selected reaction monitoring to measure protein abundance.
  2. The assays are designed and validated to target specific proteins based on their amino acid sequence.
  3. Targeting gives the assays high sensitivity in complex samples. The assays are precise, accurate, and have a wide dynamic range.
  4. Assays for individual proteins are bundled into panels of approximately 40 proteins designed to interrogate selected biochemical pathways (See Table 1).  
  5. Our workflow accommodates sufficient sample numbers to design experiments with good statistical power.
  6. Our workflow accommodates a wide range of sample types and preparation methods.
  7. We are constantly designing new methods and can design methods for new protein targets requested by users.

Description of Method.

The selected reaction monitoring (SRM) method we use is an instrumental method that is sensitive, accurate, precise, has a wide dynamic range, and high throughput.  The SRM experiment quantifies protein abundance based on the abundance of representative peptides measured in an LC-tandem mass spectrometry experiment.  The peptides are generated by a complete digestion of a protein sample using the enzyme trypsin.  The assays are designed and validated based on an extensive international database of mass spectrometry data for proteins.  Once developed, a targeted assay can be used forever and shared between laboratories.      
The assay for a protein is designed based on the amino acid sequence of the protein.  The goal of the design is to select a small number of peptides formed in the tryptic digestion that are uniquely representative of that protein and give the best response in the LC-tandem mass spectrometry experiment.  Our assay development typically begins with 6 to 10 candidates at the start and selects the best 2 peptides for the final assay.   Subsequent validation steps select the most useful transitions to monitor, optimize the collision energy, and test the assay in different types of samples.  This entire process is facilitated by reference collision induced dissociation spectra in the PeptideAtlas database.  Designing assays for proteins in different species is aided by homology to the human, mouse, rat, and bovine data present in the PeptideAtlas.  Because the key to the SRM is identifying a few that are unique peptides to a protein that can be detected by the mass spectrometer.  Therefore, small hydrophobic proteins are often difficult to analyze by the SRM method.   Figure 1 shows representative assays for two proteins detected in liver.  
 
Figure 1. Examples of the analysis of two proteins, catalase and acyl CoA oxidase, in liver.  Each protein is measured via two peptides detected in the SRM experiment.

Figure 1. Examples of the analysis of two proteins, catalase and acyl CoA oxidase, in liver. Each protein is measured via two peptides detected in the SRM experiment.

 

References:

Kinter, C.S., Lundie, J.M., Patel, H., Rindler, P.M., Szweda, L.I., and Kinter, M.  (2102) A quantitative proteomic profile of the Nrf2-mediated antioxidant response of macrophages to oxidized LDL determined by multiplexed selected reaction monitoring.  PLoS One. 7, e50016.  

Kinter, M. and Sherman, N.E. (2000) Protein Sequencing and Identification Using Tandem Mass Spectrometry.  Wiley, New York.

Kinter, M. and Kinter, C.S.  (2013) Application of Selected Reaction Monitoring to Highly Multiplexed Targeted Quantitative Proteomics: A Replacement for Western Blot.  SpringerBriefs, New York.

Ludwig, C., Claassen, M., Schmidt, A., and Aebersold, R. (2012) Estimation of absolute protein quantities of unlabeled samples by selected reaction monitoring mass spectrometry.  Mol. Cell. Proteomics. 11, M111.013987.  

Experimental Methods

Sample preparation

The protein concentration in the homogenate is measured using the BioRad detergent compatible protein assay.  This accurate protein assay is critical to the overall success of the experiment.  A volume equivalent to 60µg protein is taken and mixed with our bovine serum albumin internal standard.  The proteins are then precipitated with acetone, dissolved in 60µL Laemmli buffer and run 1.5 cm into a SDS-PAGE gel.  Each lane is cut as a single sample, the proteins reduced, alkylated, and digested with trypsin.  The final peptide mixtures are evaporated to dryness and dissolved in 150µL 1% acetic acid for analysis.   

LC-tandem mass spectrometry analysis

Our laboratory uses a ThermoScientific TSQ Vantage triple quadrupole mass spectrometry system with an Eksigent nanoflow liquid chromatography system.  Five to 10µL of the sample is injected, and the peptides eluted with a linear gradient of acetonitrile in water with 0.1% formic acid.  Retention time scheduling is used to maximize the dwell time on each transition.  Each analysis takes 80 minutes.      

Data analysis

We use the open source program Skyline to process the data (Michael MacCoss Laboratory at the University of Washington).  The initial processing finds and integrates the proper chromatographic peaks for each peptide in the panel.  The integration data are exported to Excel to complete the data reporting.  Total peptide responses are calculated as the geometric mean of the abundance of each peptide.  These responses are then normalized to bovine serum albumin peptides.

Sample Method Preparation

Sample submission

Contact Mike Kinter (mike-kinter@omrf.org) prior to submitting samples.  The goal of the contact will be to develop a detailed plan for the analyses, including: 

  • goals of the experiment,
  • experimental design including the number of samples being submitted,
  • the type and amounts of the samples that will be submitted,
  • the homogenization procedure being used, 
  • what we will do with samples 
  • time-frame for completion.

 

Sample Preparation

Our procedures require at least 150µg of protein per sample that is submitted.  Samples are submitted as homogenates that are ready for our protein assay.  Our protein assay will use approximately 1/3rd of the sample, which is required for an accurate assay of the proteins in the sample.  Samples should be clearly labeled with an informative sample name.  We will carry these names through our notes, logs, and reports.  Below are examples of methods we have the most experience using.  

 

  • Tissue samples:  The ideal sample is a cleared homogenate prepared in a standard isotonic buffer.  The buffer we routinely use is 10mM MOPS, 1mM EDTA, 210mM mannitol, 70mM sucrose, pH 7.4.  Tissue samples are homogenized with a Potter-Elvehjem homogenizer with a PTFE pestle, filtered through gauze, and cleared by homogenization at 550xg for 5min.  We use 10mL buffer per 150mg of tissue wet weight.  The final protein concentration in the homogenate is approximately 1mg/mL.  These conditions have been used for heart, liver, brain, and eye samples.  
    We have also had good success with homogenization buffers containing small amounts of detergents.  For skeletal muscle, we use 10mM MOPS, 1mM EDTA, 1% SDS, 0.5% triton, pH 7.6.  For eyes, we have used Holtz buffer. Key factors in the choice of buffers are:
    • Experience your lab has with different buffers is the best place to start.  It is generally best to continue to use the buffer system your lab has the most experience with.  We believe the combination of protein precipitation and short run gel electrophoresis makes our methods generally tolerant of the wide variety of buffer components that are available.
    • Reducing agents cannot be used.  An accurate protein assay is critical to the overall assay.  We rely on the BioRad DC (detergent compatible) protein assay, which is a variation of the Lowry assay.  It is not compatible with any reducing agents. 
    • A good understanding of the extracellular matrix in the tissue being studied is needed.  In some situations, the extracellular matrix has the potential to saturate the samples with large amounts of uninformative protein.  These types of tissues will require additional effort by the submitting lab to develop a homogenization that maximizes the extraction of cellular protein while minimizing the deleterious contribution by the extracellular matrix.  Hypotonic buffers are a possible starting point for these experiments.  
    • Consistency is critical for precise results.  Because of the potential contribution of factors like the extracellular matrix, the choice of homogenization is important.  Details like the buffer used, presence of detergents, buffer-to-tissue ratio, time, etc. can affect the comparability of the results. 
  • Cultured cells: Samples can be submitted as cell pellets harvested from at least 1million cells washed in Tris buffered saline or phosphate buffered saline.