Thorleif Lavold (Presenter)
Biomotif AB
Bio: Serial entrepreneur with long experience in the field of mass spectrometry. Also in depth knowledge of HD/Exchange applications for binding studies as well as new methods to discover the best target for potent drug candidates incl. MoA. pI Fractionation for deep proteomics and biomarker studies.
Authorship: Thorleif Lavold 2, Mohammad Pirmoradian 1,2 , Juan Astorga-Wells 1,2 , Roman A. Zubarev 1,
1 Karolinska Institutet, Stockholm, Sweden. 2 Biomotif AB, Stockholm, Sweden
| Short Abstract Online desalinator device coupled with a capillary isoelectric focusing fractionator enables direct pI-based separation of proteins in body fluids such as blood plasma. Proteomics analysis retained its high reproducibility, (median CV=3.5 %), and high correlation between the replicates (median R = 0.961). Increased depth of analysis fast, reproducible and low cost. Additional advantage is another domain for biomarker discovery, which is the difference between the positions of the same protein in patient and healthy samples representing the pI shift of the protein. |
Long Abstract
Blood plasma is the primary clinical specimen that has been studied intensively by mass
spectrometry based proteomics. The main limiting factor of biomarker discovery in blood is the dynamic range of protein concentrations stretching over 10 orders of magnitude. Consequently,method development efforts have been focused on reducing the dynamic range of samples by depletion of high-abundance proteins by immunoafinity separation, enrichment of specific protein classes (e.g. glycoprotein enrichment), as well as applying conventional fractionation methods such as gel electrophoresis, size exclusion, and reverse phase chromatography.
Today, most available procedures remain to be time-consuming, expensive and labor-intensive, in addition to sample-demanding.
Here, we present a fast, robust and sensitive fractionation method that provides additional analytical information – protein pI.
We applied the recently introduced multijunction capillary isoelectric focusing device, MJ-CIEF, for direct isoelectric separation of blood plasma samples.
To enable such separation, an online desalinator device was added that consists of a 5 cm Nafion tubular membrane with 356 μm inner diameter passed through a FEP tubing with 2 mm inner diameter. Two pooled samples - of 16 patients with progressive Alzheimer disease and of 16 healthy age-matched donors – were fractionated in five replicates each. For each replicate, 3 μL of plasma were desalinated and then pI-separated for 1 hr, and eluted into 12 fractions. The latter were individually collected, digested, and analyzed by HPLC-MS/MS via a 15 cm analytical column coupled with a Q-Exactive mass spectrometer.
Intact blood plasma samples are injected via a 6-port valve (step 1) and pass through
desalinator where buffer exchange occurs (step 2). Then the sample enters the pI-separation
column (step 3) across which electrical field is applied. After separation is completed, fractions are collected (step 4), with each fraction being digested (step 5) and purified (step 6). Each fraction is then subjected to LC-MS/MS analysis (step 7).
References & Acknowledgements:
A new publication is just accepted:
Journal: Analytical Chemistry
Title: "Multijunction capillary isoelectric focusing device combined with online membrane-assisted buffer exchanger enables isoelectric point fractionation of intact human plasma proteins for biomarker discovery"
Author(s): Pirmoradian, Mohammad; Astorga-Wells, Juan; Zubarev, Roman
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |