LC/MS-MS Quantification of Transporter Proteins

LC/MS-MS Quantification of Transporter Protein technology aims to quantify membrane protein such as ABC & SLC transporters from:

  • Microsomes or plasma membrane fraction,
  • Cell lines overexpressing transporters,
  • Organs of interest (liver, kidney, intestine, brain)

The Service is provided by SOLVO's partner: Bertin Pharma (BP). BP acquired the technology from Proteomedix Frontier (Japan), and was developed by the team of Professor Tetsuya TERASAKI from Tohoku University (Sendai, Japan).

The technology is described in detail in various publications, but can be summarized as follows:

  • Proteins are too large to be separated by reversed-phase HPLC and their mass lies above the range of the mass filter in MS for quantification.
  • Therefore, the proteins in the sample are first reduced and alkylated under solubilizing conditions in the presence of guanidine chloride or urea. The reduced and alkylated proteins are then digested with enzymes.
  • Then peptide(s) specific for the target protein can be quantified by LC/MS-MS.
  • To achieve highly selective quantification, the chosen peptide is quantified by MRM using Triple Q MS.
  • The use of two mass filters provides high selectivity and a high signal-to-noise ratio. • A stable isotope-labelled peptide having the same amino acid sequence as the target peptide is used as the internal standard.
  • Furthermore, the absolute amount of the target peptide can be calculated from the target peptide to internal standard peak ratio in chromatograms.
  • Each target peptide in a protein sample is quantified by measuring four different MRM transitions, which consist of the same Q1 and four different Q3, and the internal standard peptide is quantified by measuring the four corresponding MRM transitions.
  • Eight MRM transitions in total are required for one protein, and 37 different proteins can be simultaneously quantified by using the maximum of 300 MRM transitions in a single analysis.

A Validated technology

One of the great benefits are the validation work performed by Professor Terasaki to ensure robustness, specificity, sensitivity and monitoring of pre-analytical steps yield. From a theoretical point of view, any kind of matrix can be used. Practically, all the samples need to be cleaned, to get rid of interfering components like phospholipids. Extensive research has been performed in order to validate sample preparation, digestion and measurement of the targeted protein.

LC-MS2 selection of protein fragments - drug transporters

Sample Workflow Process

Protein concentration of the sample is measured by the Lowry method, than digested with proteases under denaturing conditions. A patented Monitoring protein is added to the sample in order to determine the purification and enzymatic digestion yield. Known amounts of the labelled peptides are spiked into the digest as internal references, and the mixture is analysed by LC/MS-MS in the MRM mode. Multiple products derived from single peptides are monitored in specific m/z channels. In the final step, individual signal peaks are identified on the basis of equal retention times in each channel of multiple product ions. To obtain the amounts of target proteins, the peptides are quantified by calculating the ratios of the peak areas to those of the isotope-labelled peptides.

Workflow LC-MS2 quantification of transporter proteins