TY - JOUR
T1 - Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
AU - Padhan, N.
AU - Yan, J.
AU - Boge, A.
AU - Scrivener, E.
AU - Birgisson, H.
AU - Zieba, A.
AU - Gullberg, M.
AU - Kamali-Moghaddam, M.
AU - Claesson-Welsh, L.
AU - Landegren, U.
PY - 2017/5/4
Y1 - 2017/5/4
N2 - Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
AB - Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
KW - Colorectal Neoplasms/diagnosis
KW - Electrophoresis, Capillary/methods
KW - Extracellular Signal-Regulated MAP Kinases/metabolism
KW - Human Umbilical Vein Endothelial Cells/metabolism
KW - Humans
KW - Isoelectric Focusing/methods
KW - Nanoparticles/chemistry
KW - Nucleic Acid Amplification Techniques/methods
KW - Phosphorylation
KW - Proteins/analysis
KW - Sensitivity and Specificity
KW - Signal Transduction/drug effects
KW - Vascular Endothelial Growth Factor A/pharmacology
UR - http://www.scopus.com/inward/record.url?scp=85018417343&partnerID=8YFLogxK
U2 - 10.1038/s41598-017-01516-7
DO - 10.1038/s41598-017-01516-7
M3 - Article
C2 - 28473697
AN - SCOPUS:85018417343
VL - 7
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 1490
ER -