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 pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp alpha-chains (Hpalpha1 and Hpalpha2) 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 alpha-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 Hpalpha1 and/or Hpalpha2 reduced intact chain(s) average mass(es). The affinity capture post-reduction approach was scaled for high-throughput Hp alpha-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.
Tubbs
Kemmons A
KA
Intrinsic Bioprobes, Inc., 625 South Smit Road, Suite 22, Tempe, AZ 85281, USA. ktubbs@intrinsicbio.com
Kiernan
Urban A
UA
Niederkofler
Eric E
EE
Nedelkov
Dobrin
D
Bieber
Allan L
AL
Nelson
Randall W
RW
D006242:Haptoglobins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007118:Immunoassay / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D010641:Phenotype; D011499:Protein Processing, Post-Translational
D006242:Haptoglobins