High-throughput MS-based protein phenotyping: Application to haptoglobin

A high‐throughput affinity capture and reduction approach was developed for phenotype and post‐translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti‐Hp antibodies immobilized onto affinity pipett...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2005-12, Vol.5 (18), p.5002-5007
Main Authors: Tubbs, Kemmons A., Kiernan, Urban A., Niederkofler, Eric E., Nedelkov, Dobrin, Bieber, Allan L., Nelson, Randall W.
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Fundamental and applied biological sciences. Psychology
Haptoglobin
Haptoglobins - chemistry
Haptoglobins - genetics
High-throughput
Humans
Immunoassay - methods
Mass Spectrometry - methods
Miscellaneous
Phenotype
Protein phenotyping
Protein Processing, Post-Translational
Proteins
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Summary:A high‐throughput affinity capture and reduction approach was developed for phenotype and post‐translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti‐Hp antibodies immobilized onto affinity pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp α‐chains (Hpα1 and Hpα2) and subjected to subsequent MALDI‐MS analysis. The affinity capture and reduction approach was originally developed from a pre‐extraction reduction methodology that was optimized to an affinity capture post‐reduction technique for intact Hp α‐chain variant analysis, phenotype classification and ensuing post‐translational variant detection. Three common Hp phenotypes (1–1, 2–1 and 2–2) were assigned according to detection of Hpα1 and/or Hpα2 reduced intact chain(s) average mass(es). The affinity capture post‐reduction approach was scaled for high‐throughput Hp α‐chain phenotype analysis from a normal plasma cohort. The entire sample cohort was successfully analyzed and phenotyped using the developed approach. Additionally, Hp post‐translational variants were detected and assigned via accurate MS analyses. The results of this study suggest use of the methodology in future analyses of other similarly complexed proteins and in normal versus disease cohort population proteomics studies.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.200500176