PMID- 32867494
OWN - NLM
STAT- MEDLINE
DCOM- 20210225
LR  - 20210225
IS  - 1520-6882 (Electronic)
IS  - 0003-2700 (Linking)
VI  - 92
IP  - 17
DP  - 2020 Sep 1
TI  - Membrane-Based Affinity Purification to Identify Target Proteins of a 
      Small-Molecule Drug.
PG  - 11912-11920
LID - 10.1021/acs.analchem.0c02316 [doi]
AB  - Identifying the target proteins of small-molecule drug candidates is important 
      for determining their molecular mechanisms of action. Porous membranes 
      derivatized with such small molecules may provide an attractive 
      target-identification platform due to a high protein-capture efficiency during 
      flow through membrane pores. This work employs carbonic anhydrase II (CAII) 
      binding to immobilized 4-(2-aminoethyl)benzenesulfonamide (AEBSA) to examine the 
      efficiency and selectivity of affinity capture in modified membranes. Selective 
      elution of captured protein, tryptic digestion, tandem mass spectrometry 
      analysis, and label-free quantification (LFQ) identify CAII as the dominant AEBSA 
      target in diluted serum or cell lysate. CAII identification relies on determining 
      the ratio of protein LFQ intensities in sample and control experiments, where 
      free AEBSA added to the control loading solution limits CAII capture. Global 
      proteomics shows that the spiked CAII is the only protein with a log(2) ratio 
      consistently >2, and the detection limit for CAII identification is 0.004 wt % of 
      the total protein in 1:4 diluted human serum or 0.024 wt % of the total protein 
      from breast cancer cell lysates. The same approach also identifies native CAII in 
      human kidney cell lysate as an AEBSA target. Comparison of affinity capture using 
      membranes, Affi-Gel 10 resin or M-270 Dynabeads derivatized with AEBSA suggests 
      that only membranes allow identification of low-abundance CAII as a target.
FAU - Yang, Liu
AU  - Yang L
FAU - Bui, Loan
AU  - Bui L
FAU - Hanjaya-Putra, Donny
AU  - Hanjaya-Putra D
FAU - Bruening, Merlin L
AU  - Bruening ML
AUID- ORCID: 0000-0002-4553-5143
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20200811
PL  - United States
TA  - Anal Chem
JT  - Analytical chemistry
JID - 0370536
SB  - IM
MH  - Chromatography, Affinity/*methods
MH  - Humans
MH  - Membranes/*metabolism
MH  - Protein Binding/*physiology
EDAT- 2020/09/02 06:00
MHDA- 2021/02/26 06:00
CRDT- 2020/09/02 06:00
PHST- 2020/09/02 06:00 [entrez]
PHST- 2020/09/02 06:00 [pubmed]
PHST- 2021/02/26 06:00 [medline]
AID - 10.1021/acs.analchem.0c02316 [doi]
PST - ppublish
SO  - Anal Chem. 2020 Sep 1;92(17):11912-11920. doi: 10.1021/acs.analchem.0c02316. Epub 
      2020 Aug 11.