Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation

Abstract

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.Capillary electrophoresis (CE), coupled with fluorescence detection, liquid chromatography or mass spectrometry, has many applications in separating and detecting biomolecules such as DNA 1 , metabolites 2 , peptides 3 and proteins 4 , since it offers high resolution and uses small amounts of samples. In particular, capillary isoelectric focusing (IEF) has proven useful for resolving protein isoforms such as phosphorylated variants 5 , since they typically have different isoelectric points (pI), allowing their separation in an ampholyte gradient gel.The NanoPro 1000 system from ProteinSimple is a recently developed automated capillary IEF system where proteins are first separated and then immobilized in capillaries, followed by antibody probing in analogy to standard immunoblotting for protein identification and quantification 6 . However, the system differs from immunoblotting in two important respects 7 : First, in conventional immunoblotting proteins are separated according to molecular weight, while separation by IEF depends on the charge of the investigated protein species. Secondly, in immunoblotting, proteins are denatured by treatment with an ionic detergent while proteins separated by capillary IEF remain in a native state, which may influence the ability of antibodies to recognize the proteins. The capillary IEF method permits the use of limited tissue samples, as the technique efficiently resolves and quantifies proteins and their isoforms in submicroliter samples. Native proteins extracted from lysates of cells or tissues are separated according to charge, resolving isoforms of the proteins, whereupon the proteins are immobilized on

Potency Assessment of CBD Oils by Their Effects on Cell Signaling Pathways

“…In this study, we showed that multiplexed immunoassays in nanocapillaries permitted simultaneous analysis of nine signaling pathways in cultured SH-SY5Y cells. Notably, nanofluidic protein PTM profiling is ideally suited for applications on clinical specimens due to its ultrasensitivity, where nanograms of tissue lysates are sufficient for the detection of picograms of proteins of interest [12,13,21,52]. Furthermore, high reproducibility between measurements, reliable quantitation, and low user errors due to automated operation are among the attributes that highlight the robustness of the technology [25,53].…”

Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

“…The in situ PLA technique first presented in 2006 5 , the proximity hybridization chain reaction (ProxHCR) 28 , and with this paper now also UnFold in situ PLA reactions. All these three techniques share the advantage that cross-reactive detection of irrelevant proteins is less likely since only targets recognized by pairs of antibodies can give rise to detection signals, greatly reducing risks of nonspecific background 29 , 30 . The three techniques are also all useful to investigate interactions or close proximity between pairs of proteins where each protein is bound by one antibody, and the same principle also holds for detection of posttranslational modifications, where one antibody is devoted to detection of the protein and another recognizes the modification, as illustrated herein for protein phosphorylations.…”

Chaperome heterogeneity and its implications for cancer study and treatment

“…1) (46). Based on the NanoPro 1000 system from ProteinSimple (135), it uses an immobilized pH gradient to separate native multimeric protein complexes based on their pI and allows for subsequent probing of immobilized complexes with native cognate antibodies. Proteins separated by capillary IEF remain in a native state, which may influence the ability of antibodies to recognize the proteins, and thus we screened a panel of antibodies to identify those that best capture the epichaperome complexes.…”