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Depletion of high-abundance proteins from serum by immunoaffinity chromatography: a MALDI-FT-MS study
Lennard J Dekker1, Jan Bosman, Peter C Burgers
1Department of Neurology, ErasmusMC, Rotterdam, The Netherlands.
Insights
High-abundance protein immunodepletion from serum is reproducible for biomarker discovery. This study confirmed consistent results across 250 prostate cancer serum samples over six weeks using immunoaffinity depletion.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Immunodepletion of high-abundance serum proteins is crucial for biomarker discovery.
- Ensuring reproducibility of this depletion step is vital for reliable study outcomes.
Purpose of the Study:
- To assess the reproducibility of serum protein immunodepletion.
- To evaluate the consistency of the depletion process over an extended period (6 weeks) and across numerous samples (250).
Main Methods:
- Utilized immunoaffinity depletion on a single column for 250 prostate cancer serum samples over 6 weeks.
- Employed automated peak detection and fraction collection, analyzing reproducibility via 280nm peak surface area and fraction volume.
- Assessed depletion efficiency of key proteins (albumin, transferrin, alpha-1-antitrypsin) and remaining proteins (apolipoprotein A1) using MALDI-FT-MS.
Main Results:
- Depletion reproducibility showed <7% relative standard deviation (R.S.D.) for peak surface area.
- Collected fraction volume was consistent at 0.97mL (4.5% R.S.D.).
- Demonstrated significant reduction of target high-abundance proteins and a notable increase in the relative abundance of apolipoprotein A1 post-depletion.
Conclusions:
- The immunodepletion process for high-abundance serum proteins is highly reproducible.
- The method ensures consistent results over a 6-week period, supporting its reliability in biomarker discovery studies.
- The depletion was found to be complete for the targeted proteins.
Abstract:
Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-FT-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks.
