An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases

Abstract

Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-β family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.

Fig. 1.

MIB-PRM workflow. Undigested protein lysate is passed through a gravity layered multidrug inhibitor beads column. Broad classes of activated kinases are preferentially enriched in the eluate. A tryptic digest of the eluate is loaded on a meter-scale monolithic silica-C18 column and resolved using a 6-h reverse phase chromatography gradient at a constant temperature of 40 °C. Online targeted detection of endogenous kinases by a highly multiplexed label-free PRM method provides high sensitivity and specificity of detection compatible with automated data processing.

Fig. 2.

Performance characteristics of a meter-scale monolithic silica-C18 column desirable for targeted proteomic applications. Tryptic digest of mouse bone marrow lysate was separated on the 200-cm monolithic silica-C18 column at 25, 40, 60, and 80 °C and analyzed by LC-MS/MS. A, Peak width as a function of gradient time is calculated as a running median of full width at half maximum. B, Peak capacity as a function of gradient time is calculated as the running median of peak width divided by gradient duration. C, Number of peptides and proteins identified at each temperature. D, Triplicate runs of the mouse bone marrow lysate sample were performed on the 200 cm monolithic silica-C18 column (at 40 °C) and 50 cm C18 silica particle-packed column (at 45 °C). Histogram of RT standard deviations for peptides identified in all replicates is plotted.

Fig. 3.

Highly multiplexed label-free PRM is achieved with long gradient chromatography on meter-scale monolithic silica-C18 column. 1067 peptides from 325 mouse proteins belonging to GO category “Cellular Process” were included in a single PRM method utilizing a 6-h reverse phase chromatography gradient on the 200-cm monolithic silica-C18 column. Tryptic digest of the mouse bone marrow lysate was used to test the method. Data were analyzed in Skyline using automated peak detection. A, Scheduling diagram shows the expected maximum number of targets as a function of time along the gradient based on the overlap of target detection windows. B, Distributions of library dot product and peaks found ratio, two metrics of detection specificity calculated by Skyline, are shown as box plots (solid line = median, box = interquartile range, whiskers = 1.5 × interquartile distance, dots = individual observations outside whiskers). C, Histogram of MS2 fragment mass accuracy in parts per million (ppm) calculated by Skyline. D–F, Specificity of detection in relationship to protein abundance is estimated by library dot product (D), MS2 fragment mass accuracy (E), and peaks found ratio (F) as a function of peak area.

Fig. 4.

Three types of selective inhibitors of MAPK signaling produce expected differential kinase inhibition and activation responses in HCT116 colorectal cancer cells. HCT116 cells were treated with 250 nm of GDC-0973, GDC-0623, SCH772984 or DMSO for 1, 4, or 24 h. In the washout samples, cells were drug treated for 24 h, then changed into fresh media and harvested after 0.5 or 2 h. Lysates were used for Western blots of total and phosphorylated MEK, ERK, and RSK; blotting for COX IV was used as the loading control.

Fig. 5.

Highly multiplexed PRM method downstream of MIB kinase enrichment quantifies kinome response to three types of MAPK signaling inhibitors in HCT116 colorectal cancer cells. HCT116 cells were treated with 250 nm GDC-0973, GDC-0623, SCH772984, or DMSO for 90 min. The lysates were collected and passed over MIB columns. The eluates enriched for activated kinases were digested with trypsin and analyzed by a single PRM method utilizing the long monolithic silica-C18 column and targeting 929 precursor peptides from 182 kinases. A total of 814 precursors from 154 kinases were quantified using strict specificity criteria. A, Abundance of MAPK1 (ERK2), MAPK3 (ERK1) and MAP2K1 (MEK1) in MIB eluates measured by PRM are plotted as log2 ratios to DMSO control. B, All kinases with statistically significant inhibition or activation relative to DMSO control were ordered by hierarchical clustering of the corresponding log2 ratios. C, HCT116 cells were treated with 250 nm of GDC-0973, GDC-0623, SCH772984, or DMSO for 90 min, or with TGF-β at 50 pg/ml for 30 min, followed by Western blotting.