Antibodies that recognize PTMs of histones play a central role in epigenetic proteomic research. Modification-specific antibodies are employed in chromatin immunoprecipitation, for Western blotting and during the immunoprecipitation steps for MS-based global proteomic analyses. Knowledge about the antibodies' off-target binding is essential for the interpretation of experimental data. To address this challenge we developed a fast and cost efficient system for generating peptide bead arrays. We employed this method to establish a bead-based peptide array containing 384 peptides displaying phosphorylated, acetylated, methylated, and citrullinated N-terminal regions of histones H2A, H2B, H3 and H4 and controls. We profiled the binding of 40 PTM-specific antibodies important for epigenetic proteomic research.
Multitransmembrane proteins are notoriously difficult to analyze. To date, rapid, and cost-efficient detection methods are lacking and only mass spectrometry-based systems allow reliable quantification of these proteins. Here, we present a novel type of sandwich immunoassay that is capable of sensitively detecting multidrug resistance protein 1 (MDR1), a prototypic 12-transmembrane-domains transporter. In a first assay step, complex samples are enzymatically fragmented into peptides as routinely done for mass spectrometry. A proteotypic peptide derived from MDR1 was chosen and antibodies targeting this peptide were used to build a sandwich immunoassay. Validation of the optimized assay showed good sensitivity, reproducibility and it allowed reliable quantification of MDR1; cross-validation by mass spectrometry demonstrated the applicability for routine analyses in clinical and pharmaceutical research. MDR1 was quantified in primary human renal cell carcinoma and corresponding normal tissue and down-regulation or expression loss was found in tumor tissue corroborating its importance in drug resistance and efficacy.
Macrophage colony stimulating factor 1 receptor (MCSF1R), osteopontin
(OPN), high-mobility group protein B1 (HMGB1), glutamate dehydrogenase
(GLDH), keratin 18 (K18), and caspase-cleaved keratin 18 (ccK18) are
considered promising mechanistic biomarkers for the diagnosis of drug-induced
liver injury. Here, we aim to elucidate the impact of the sample matrix
and handling on the quantification of these emerging protein biomarkers.
We investigated effects such as time from collection to centrifugation
during serum (± gel) or EDTA plasma preparation on two assay
platforms: immunoaffinity liquid chromatography mass spectrometric
assays and sandwich immunoassays. Furthermore, we measured GLDH activity
with an enzymatic activity assay. Matrix effects were observed particularly
for HMGB1 and MCSF1R. HMGB1 levels were higher in serum than in plasma,
whereas higher concentrations of MCSF1R were observed in plasma than
in serum. A comparison of sample collection to centrifugation time ranging from 15 to 60 min demonstrated
increasing levels of HMGB1 in serum, while MCSF1R, OPN, GLDH, and
ccK18 concentrations remained stable. Additionally, there was a poor
correlation in HMGB1 and ccK18 levels between serum and plasma. Considering
the observed matrix effects, we recommend plasma as a matrix of choice
and cross-study comparison studies to be limited to those using the
same matrix.
A system-wide analysis of cell signaling involves detecting and quantifying a range of proteins and their posttranslational modification states in the same cellular sample. We propose a protocol for a miniaturized, bead-based array and describe its efficiency in characterizing the different forms and functions of β-catenin. The protocol provides detailed instructions for cell culture and bead array assays that enable insights into complex networks at the systems level.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.