Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides

Abstract

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease.

Target Identification in Small Cell Lung Cancer via Integrated Phenotypic Screening and Activity-Based Protein Profiling

“…The intensity of certain peptides could be too weak to be detected regularly, particularly as covalent modification with ATP-desthiobiotin may negatively impact peptide identification by MS. Migrating from LC-MS/MS to targeted mass spectrometry (multiple reaction monitoring) can make peptide analyses ( i.e. fewer missing values) and quantification more precise (4, 5, 30). To this end, we developed an ABPP-LC-multiple reaction monitoring approach that can enable rapid and reproducible detection as well as quantified measurements of peptides corresponding to other panels of enzymes in cells and tissues that could be used for additional target validation following screening (30).…”

“…fewer missing values) and quantification more precise (4, 5, 30). To this end, we developed an ABPP-LC-multiple reaction monitoring approach that can enable rapid and reproducible detection as well as quantified measurements of peptides corresponding to other panels of enzymes in cells and tissues that could be used for additional target validation following screening (30). Fourth, while desthiobiotin-ATP probes are designed to engage the ATP binding site of proteins, we have observed recovery of peptides from outside the ATP binding pocket ( e.g.…”

Activity-Based Protein Profiling Shows Heterogeneous Signaling Adaptations to BRAF Inhibition

“…(36) Briefly, cells were solubilized in 400 μl of lysis buffer and 4 μl of Halt™ phosphatase and protease inhibitor cocktail (Catalog #78440, Thermo). Samples were sonicated thrice at 1-minute intervals using a pulse of 30% duty cycle for 30 seconds (Cell Disruptor 200, Branson, Danbury, CT).…”

Introduction for the Special Issue of METHODS on Detection and quantification of proteins in clinical samples by mass spectrometry

“…c o m / l o c a t e / y m e t h leukemia cells. In the methods described by Fang et al [5], hundreds of desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are quantified in single LC-MRM experiments in lung cancer cell lines and tissues to evaluate their kinome. The work of Zhang et al [6] presents a sample preparation method and LC-MS/MS settings that enable the proteome analysis of optimal cutting temperature (OCT)-embedded primary patient tissue, applied to non-small cell lung carcinoma.…”

“…The second paper, by Gallien and Domon [2], discusses in detail the different essential steps for the development of a quantitative proteomics parallel reaction monitoring (PRM) experiment, a technique characterized by higher selectivity and confidence level in target assignment that are allowed by the recently developed mass spectrometers capable of high-resolution and accurate-mass (HR/AM) measurements. The following five articles discuss different quantitative proteomics methods applied to cancer biology [3][4][5][6][7]. Percy et al [3] describe a method for the multiplexed measurement of a large panel (>100) of protein biomarkers in urine samples using the mass spectrometry mode of multiple reaction monitoring (MRM) with stable isotope-labeled standards (SIS) for internal normalization, which was applied to a patient cohort with prostate cancer.…”