Multiplexed quantification of plant thylakoid proteins on western blots using lanthanide-labeled antibodies and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

TY - JOUR

T1 - Multiplexed quantification of plant thylakoid proteins on western blots using lanthanide-labeled antibodies and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

AU - de Bang, Thomas Christian

AU - Pedas, Pai

AU - Schjørring, Jan Kofod

AU - Jensen, Poul Erik

AU - Husted, Søren

PY - 2013/5/21

Y1 - 2013/5/21

N2 - We have developed a novel calibration method that allows concurrent quantification of multiple proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after Western blotting. Calibrants were made of nitrocellulose membranes doped with lanthanide standards. Excellent linearity was obtained in the interval from 0 to 24 ng lanthanide cm-2. Cerium-labeled lysozyme was introduced as an internal reference protein, enabling correction for up to 50% difference in transfer efficiency during the blotting of membranes. The sensitivity of the LA-ICP-MS method was comparable to state-of-the-art chemiluminescence detection and was further improved by a factor of 20, using a polymer tag. Our method allowed reproducible and multiplexed quantification of five thylakoid proteins extracted from chloroplasts of the plant species Arabidopsis thaliana (relative standard deviation (RSD) of ≤ 5% in three independent analytical series). The method was capable of measuring the L subunit in photosystem I of an Arabidopsis mutant containing <5% of this particular protein, relative to the wild type. We conclude that the developed calibration method is highly suited for multiplexed and comparative protein studies, allowing for intermembrane comparisons with high sensitivity and reproducibility.

AB - We have developed a novel calibration method that allows concurrent quantification of multiple proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after Western blotting. Calibrants were made of nitrocellulose membranes doped with lanthanide standards. Excellent linearity was obtained in the interval from 0 to 24 ng lanthanide cm-2. Cerium-labeled lysozyme was introduced as an internal reference protein, enabling correction for up to 50% difference in transfer efficiency during the blotting of membranes. The sensitivity of the LA-ICP-MS method was comparable to state-of-the-art chemiluminescence detection and was further improved by a factor of 20, using a polymer tag. Our method allowed reproducible and multiplexed quantification of five thylakoid proteins extracted from chloroplasts of the plant species Arabidopsis thaliana (relative standard deviation (RSD) of ≤ 5% in three independent analytical series). The method was capable of measuring the L subunit in photosystem I of an Arabidopsis mutant containing <5% of this particular protein, relative to the wild type. We conclude that the developed calibration method is highly suited for multiplexed and comparative protein studies, allowing for intermembrane comparisons with high sensitivity and reproducibility.

U2 - 10.1021/ac400561q

DO - 10.1021/ac400561q

M3 - Journal article

C2 - 23593924

SN - 0003-2700

VL - 85

SP - 5047

EP - 5054

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

IS - 10

ER -