Quantitation Options

Quantitation

Whilst qualitatively profiling the protein content of a complex may be sufficient for many studies, it is often required that the relative levels of complex members be monitored across several experiments, for example when looking at separate pull downs of each member of a complex. We are able to efficiently determine relative levels by following the same GeLC/MS workflow for each sample and employing spectral counting to perform quantitation. Here, the number of peptides matching to a protein and, importantly, the number of times those peptides are observed (spectral count) allows inference of protein abundance between samples. The use of peptide internal standards allows us to measure absolute levels of each protein and therefore infer stoichiometric information about members of a complex.

Protein Quantitation

Our quantitative proteomic workflows are also utilized in the discoveryexpress service and involve either labeled or non-labeled approaches. An overview of the options available with benefits/features is given here:

GeLC-MS

Features Process
  • Applicable to most biological matrices (cells, tissues, biofluids, spent media (secretome), plants)
  • High number of proteins detected (up to 4,000 in cell lines) and high protein coverage
  • Provides information-rich proteome maps including isoforms present
  • Quantitation information on every protein identified
  • Low sample requirements (1-20µg protein)
  1. Run each sample on a lane of an SDS-PAGE gel
  2. Segment each lane (10, 24, 40 segments depending on sample complexity)
  3. In-gel digestion of proteins
  4. Mass spectrometry (LC-MS/MS) for identification and quantification of proteins
  5. Data analysis, statistics

Differential mass spectrometry (dMS, label-free mass spectrometry)

Features Process
  • High throughput
  • Amenable to large number of samples
  • Focused on differentially expressed proteins
  • Fractionation provides higher number of proteins with a lower throughput
  • Amenable to non-sequenced genomes
  1. Sample preparation, optional fractionation step, protein digestion
  2. Mass spectrometry (LC-MS/MS)
  3. Alignment of peptide chromatographic peaks
  4. Calculation of relative quantification using peak areas
  5. Identification of differentially expressed proteins

SILAC


www.silac.org
Features Process
  • Pair wise comparison of samples
  • High number of proteins detected and quantified
  1. Cells grown in medium containing heavy amino acids to label proteins
  2. Sample preparation and fractionation
  3. Protein digestion and mass spectrometry (LC-MS/MS)
  4. Data analysis

Global phosphoprotein analysis

Features Process
  • Detection and quantification of phosphorylated proteins
  1. Sample preparation
  2. Enrichment of phosphoproteins or phosphopeptides with antibodies and/or capture reagents
  3. Mass spectrometry (LC-MS/MS) analysis using dMS
  4. Data analysis

Targeted Analysis

Features Process
  • Detection and quantification of known peptides using LC-MRM/MS
  1. Sample preparation:
    • Protein depletion (optional)
    • Protein fractionation (optional)
    • Protein enrichment (optional)
  2. Protein digestion
  3. Mass spectrometry (LC-MRM/MS):
    • Relative quantitation
    • Absolute quantitation (requires labelled internal standards)
  4. Data analysis