Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

Abstract

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.

Direct Quantification of Cytochromes P450 and Drug Transporters—A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates

“…Mobile phase A was composed of 99.9% double distilled H 2 O and 0.1% FA, and mobile phase B was composed of 80% acetonitrile with 0.1% FA. Sample measurement using a full-MS method on a Q Exactive Plus (Thermo Scientific) and data analysis were performed as described previously (Weiß et al, 2015).…”

“…More recently, we introduced peptide group-specific antibodies [triple X proteomics (TXP) antibodies] to this workflow. These antibodies bind short C-terminal sequences of proteotypic tryptic peptides, allowing peptides to be enriched with high sequence similarities using just a single antibody for several analytes (Weiß et al, 2015;Groll et al, 2016;Marx-Stoelting et al, 2017). This method was used to analyze P450 and transporter proteins directly from cell culture or tissue samples without any prefractionation steps, such as membrane enrichment (Kawakami et al, 2011;Ohtsuki et al, 2012;Schaefer et al, 2012) or SDS-PAGE (Langenfeld et al, 2009;Miliotis et al, 2011).…”

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…Sample preparation can also contribute to this variability, with proteins either quantified directly in whole cell/tissue lysate (Weiß et al, 2015;Wi sniewski et al, 2016) or in enriched subcellular fractions Gröer et al, 2013). In addition, differences in proteolytic strategies, efficiency of protein and peptide recovery and LC-MS analysis of peptides have also been suggested to introduce bias into abundance measurements (Chiva et al, 2014;Harwood et al, 2015).…”

Toward a Consensus on Applying Quantitative Liquid Chromatography‐Tandem Mass Spectrometry Proteomics in Translational Pharmacology Research: A White Paper

“…Immuno‐enrichment is not inherently capable of distinguishing between intracellular and plasma membrane proteins and adding a biotinylation step to this method could theoretically combine the advantages of the two techniques. In cell lysates, the lower reproducibility observed with the lower range of protein expression can be addressed by immuno‐enrichment of peptides from these targets; however, generating antibodies for all required targets with sufficient specificity remains a complex challenge, leading to limited proteome coverage . Multiplexing is also challenging with these technologies and the presence of protein variants (e.g., SNPs and PTMs) can lead to changes in the dynamics of interaction between the antibodies and their target analytes.…”

“…42 Enrichment with antibodies can STATE of the ART take place at the protein level, 43 with applications in elucidating transcription regulatory networks, cell signaling pathways, and biomarker discovery, or postdigestion at the peptide level for peptide enrichment before LC-MS/MS. 42 An example of such technique is Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA), also referred to as immunoselected reaction monitoring technology. 43 This approach allows reduction of matrix complexity within a whole-cell digest by enriching only the desired peptides, followed by MS analysis.…”