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Title: Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: Analysis of human plasma and cerebrospinal fluid

Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 µL) due to low yields stemming from losses caused by nonspecific binding to the column matrix. Additionally, the cost of the depletion media can be prohibitive for larger scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large scale studies. We characterized the performance of a 346 µL column volume micro-scale depletion system, using four different flow rates to determine the most effective depletion conditions for ~6 μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10 mL depletion column served as the control. Results showed depletion efficiency of the micro-scale column increased as flow rate decreased, and that our micro-depletion was reproducible. We found, in an initial application,more » a 600 µL sample of human cerebral spinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.« less
 [1] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Korea Research Inst. of Standards and Science (Korea)
  2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Publication Date:
Report Number(s):
Journal ID: ISSN 1618-2642; 47418; 40072; 48135; 400412000
Grant/Contract Number:
AC05-76RL01830; AC05-76RLO01830; 8 P41 GM103493-10; 5 P41 RR018522-10
Accepted Manuscript
Journal Name:
Analytical and Bioanalytical Chemistry
Additional Journal Information:
Journal Volume: 406; Journal Issue: 28; Journal ID: ISSN 1618-2642
Research Org:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Sponsoring Org:
Country of Publication:
United States
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; microscale depletion; IgY-14 immunoaffinity resin; Human plasma; cerebrospinal fluid; MS; Environmental Molecular Sciences Laboratory
OSTI Identifier: