A cross-species proteomic assessment of cost-effective platforms for depleting high-abundant proteins from blood serum

02 June 2025, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Blood proteome analysis is becoming increasingly popular in veterinary research because many animal models have been used to study a range of human diseases. Most of the commercial high-abundance protein (HAP) depletion kits are optimized for human serum, whereas proteins in animal serum may not be present in human serum or may be present at different abundances. There are no previous studies investigating the efficacy of using various HAP kits for proteome analysis of animal serums. We used three commercial serum abundant protein depletion (SAPD) kits (i.e., ion exchange-based Norgen kit (ProteoSpin™), antibody-based Thermo Albumin Depletion Kit (Pierce™), solubility-based kit (Minute™), and a cost-effective method (i.e., perchloric acid (PerCA) HAP precipitation) to assess their effectiveness to process serums from five different species (i.e., mouse, chicken, dog, goat, and guinea pig). Protocols of the commercial kits were adopted from manufacturers' guidelines with minor modifications for optimized performance. Following HAP depletion, proteins from all species were digested using a Trypsin/Lys-C enzyme mix, desalted, and subjected to label-free quantitative bottom-up proteomics analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The raw data were processed using the Andromeda search engine integrated into MaxQuant, and peptide identification was performed by searching against the UniProt-reviewed protein database. Advanced bioinformatics tools were employed to facilitate data analysis and visualization, ensuring comprehensive interpretation of the depletion efficiency and comparative performance of the methods across species. We determined their capabilities of protein identification (Norgen kit > Minute kit > PerCA precipitation > Thermo kit), depletion efficiencies of HAPs (Minute kit > Norgen kit > PerCA precipitation > Thermo kit), and cost-effectiveness (PerCA precipitation > Minute kit > Norgen kit > Thermo kit). Our results show that the PerCA precipitation method, which is >20 times cheaper than commercial kits, outperforms other methods in depleting HAPs, especially in mouse serum. While Norgen kit excels in mouse and goat serum, the PerCA precipitation method offers broader applicability and reveals unique low abundant proteins. Protein pathway analysis highlights distinct biological processes affected by different depletion methods. Overall, our studies provide valuable insights into protein depletion techniques, with the PerCA depletion method emerging as a cost-effective and versatile option for proteomics research across various serums.

Keywords

Cross species
High abundance protein
Depletion methods
Proteomics

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.