Recent instrumentation and bioinformatics developments have led to new quantitative mass spectrometry platforms including LC-MS/MS with data-independent acquisition (DIA) and targeted analysis using parallel reaction monitoring mass spectrometry (LC-PRM), providing alternatives to well-established methods, such as LC-MS/MS with data dependent acquisition (DDA) and targeted analysis using multiple reaction monitoring mass spectrometry (LC-MRM). These tools have been used to identify signaling perturbations in lung cancers and other malignancies, supporting the development of effective kinase inhibitors and, more recently, provided insights on drug resistance mechanisms and repurposing opportunities. However, detection of kinases can be challenging; therefore, activity-based protein profiling used to enrich for ATP-utilizing proteins was selected as a test case for exploring the limits of detection of low abundance analytes in complex biological samples. To examine the impact of different MS acquisition platforms, quantification of kinase ATP-uptake following kinase inhibitor treatment was analyzed by four different methods: LC-MS/MS with DDA and DIA, LC-MRM, and LC-PRM. For discovery datasets, DIA increased the number of identified kinases by 21% and reduced missingness when compared to DDA. In this context, MRM and PRM were most effective at identifying global kinome responses to inhibitor treatment, highlighting the value of a priori target identification and manual evaluation of proteomics datasets. We show comparable results for a selected set of desthiobiotinylated peptides from PRM, MRM, and DIA and identify considerations for selecting a quantification method and post-processing steps that should be used depending on the acquisition method employed.

All samples were separated by reversed phase nano-liquid chromatography (Dionex RSLC, Thermo) and analyzed by MRM or PRM (DDA and DIA acquisitions available on PRIDE). Each condition was analyzed with the four LC-MS methods a total of three times from two biological replicates. For PRM analysis, peptides were separated on a 90 minute linear gradient from 5 – 35% B Solvent (90% ACN containing 0.1% formic acid) in A solvent on a C18 column, 75 µM x 500 mm (Thermo, Acclaim Pepmap). Prior to MRM acquisition, peptides were separated on a 90 minute gradient from 5 – 35% B solvent on a C18 column, 75 µM x 250 mm (Thermo, Acclaim Pepmap). MRM analysis was performed on a TSQ Quantiva (Thermo) with a capillary temperature of 275° C and a spray voltage of 2100 V. The Q1 and Q3 resolution values were set to 0.4 and 0.7, respectively, with a dwell time of 10 ms per transition. A method containing 1,627 transitions corresponding to 409 desthiobiotin-labeled peptides was scheduled using iRT. Variable retention time windows were used, with the center of the gradient utilizing 10 minute acquisition windows. Collision energy (CE) values were calculated using CE optimization equations empirically derived with previous datasets in Skyline. PRM samples were analyzed using a Q-Exactive Plus MS with a capillary temperature of 250° C and spray voltage of 2400 V. PRM analysis used the same kinase desthiobiotin-labeled peptide panel as MRM. To reduce crowding, PRM retention time windows were set to 8 minutes around the expected precursor detection time. PRM analysis did not include MS1 data acquisition, and MS/MS resolution was 17,500.

NCI-H1993 cells were obtained from the Moffitt Cancer Center Lung Cancer Center of Excellence Cell Core, where they have been authenticated with short tandem repeat analysis and are routinely tested for mycoplasma contamination. Cells were grown in RPMI-1640 media containing L-glutamine and HEPES (Gibco), supplemented with 10% fetal bovine serum (Hyclone), 10 U/mL Penicillin/Streptomycin (Gibco), and 2.5 g/L glucose (Sigma). Cells were grown to 70% confluence for about 24 hours, and then treated with DMSO, 200 nM BEZ-235, or 500 nM Crizotinib (Selleck Chemicals) for 24 hours before harvesting. Plates were washed twice with ice cold PBS containing 1 mM Sodium orthovanadate (Sigma) prior to scraping the cells off the culture dish in 600 µL of Pierce IP Lysis Buffer containing protease inhibitors (Thermo). Cell extracts were sonicated on ice and cleared by centrifugation at 18,000 x g for 20 minutes at 4° C prior to desalting using Zeba Spin Columns (Thermo). The protein concentration in each lysate was determined using Bradford Assays (Coommassie Plus Protein Assay, Thermo). Cell lysates were labeled for activity-based protein profiling (ABPP) following the manufacturer’s recommendations for the Pierce Kinase Enrichment Kit with ActivX Probes (Thermo). Briefly, 1 mg of total protein from each desalted lysate was used in quadruplicate for each treatment condition. Following incubation with 20 mM MnCl2 for 5 minutes, lysates from cells (from the vehicle controls described above) were treated with DMSO, Dasatinib, or Erlotinib (Selleck Chemi-cals) at a final concentration of 10 µM for 10 minutes. Lysates were then incubated with 10 µM desthiobiotin-ATP probe for an additional 10 minutes. All incubation steps were performed at room temperature. Labeled lysates were denatured in 5 M urea and 5 mM DTT for 30 minutes at 65° C, and then alkylated with 40 mM iodoacetamide (Sigma). Samples were desalted again into a HEPES digestion buffer and digested overnight with 20 µg Trypsin (Worthington) at 37° C. Digested lysates were incubated for 2 hours at room temperature with high-capacity streptavidin beads to enrich for desthiobiotinylated peptides. Beads were sequentially washed multiple times with lysis buffer, 1x PBS, and water; labeled peptides were eluted using aqueous 50% acetonitrile (ACN) with 0.1% TFA. Eluted peptides were vacuum concentrated (SpeedVac, Thermo) and reconstituted in 20 µL of A solvent (2% ACN with 0.1% formic acid) containing 1 fmol/µL Pierce Retention Time Calibrator (PRTC) standard peptides (Thermo).