Supporting Information for “A High-Throughput Targeted Proteomic Approach for Comprehensive
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Supporting Information for “A High-throughput Targeted Proteomic Approach for Comprehensive Profiling of Methylglyoxal-induced Perturbations of Human Kinome” Weili Miao, Yongsheng Xiao, Lei Guo, Xiaogang Jiang, Ming Huang and Yinsheng Wang* Correspondence author: Yinsheng Wang. Telephone: (951)827-2700; E-mail: [email protected] (Anal. Chem., 2016) S1 Materials and Methods The desthiobiotinylated nucleotide affinity probes were prepared according to previously published procedures.1,2 Labeling of ATP-binding proteins, tryptic digestion, and affinity purification of desthiobiotin-C3-labeled peptides Approximately 2×107 cells were harvested, washed with cold PBS for three times, and lysed in a 1 mL lysis buffer, which contained 0.7% CHAPS, 50 mM HEPES (pH 7.4), 0.5 mM EDTA, 100 mM NaCl, and 10 µL (1:100) protease inhibitor cocktail on ice for 30 min. The cell lysates were centrifuged at 16,000g at 4°C for 30 min and the resulting supernatants were collected. Endogenous nucleotides in the resulting protein extract were removed by gel filtration using a NAP-25 column (Amersham Biosciences). Cell lysates were subsequently eluted into a buffer containing 50 mM HEPES (pH 7.4), 75 mM NaCl, and 5% glycerol. The amounts of proteins in the lysates were quantified using Quick Start Bradford Protein Assay (Bio-Rad). Prior to the labeling reaction, MgCl2, MnCl2, and CaCl2 were added to the concentrated cell lysate until their final concentrations reached 50, 5 and 5 mM, respectively. Approximately 1.0 mg cell lysate in a 1.0-mL solution was treated with the light desthiobiotin-C3-ATP affinity probe (Figure 1a) at a final concentration of 100 µM. Labeling reactions were conducted at room temperature with gentle shaking for 2.5 h. After the reaction, the remaining probes in the cell lysates were removed by buffer exchange with 50 mM NH4HCO3 (pH 8.5) using Amicon Ultra-4 filters (10,000 NMWL, Millipore). After addition of 8 M urea for protein denaturation, dithiothreitol and iodoacetamide for cysteine reduction and alkylation, the labeled proteins were digested with modified sequencing-grade trypsin (Roche Applied Science) at an enzyme/substrate ratio of 1:100 in 50 mM NH4HCO3 (pH 8.5) at 37°C overnight. The peptide mixture was subsequently dried in a Speed-vac and redissolved in 1.0 mL PBS buffer (100 mM potassium phosphate and 0.15 M NaCl, pH 7.5), to which solution was subsequently added 300 µL avidin-agarose resin (Sigma-Aldrich). The mixture was then incubated at room temperature for 1 h with gentle shaking. The agarose resin was washed sequentially with 3 mL PBS buffer and 3 mL H2O to remove unbound peptides, and the desthiobiotin-conjugated peptides were subsequently eluted with 1% TFA in CH3CN/H2O (7:3, v/v) at 75°C. The resulting enriched peptide samples were desalted by employing OMIX C18 S2 pipet tips (Agilent Technologies) and analyzed on an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) for discovery-mode analysis. LC-MS/MS analysis on an LTQ-Orbitrap Velos mass spectrometer Probe pull-down samples were analyzed by on-line 2D-LC-MS/MS and synthetic peptides were analyzed by 1D-LC-MS/MS on an LTQ-Orbitrap Velos mass spectrometer equipped with a nanoelectrospray ionization source. The fully automated 8-cycle on-line two-dimensional LC- MS/MS was set up as described3 with minor modifications. Briefly, samples were automatically loaded from a 48-well microplate autosampler using an EASY-nLC II system (Thermo Fisher Scientific) at 3 µL/min onto a biphasic precolumn (150 µm i.d.) comprised of a 3.5-cm column packed with 5 µm C18 120 Å reversed-phase material (ReproSil-Pur 120 C18-AQ, Dr. Maisch) for 1D-LC-MS/MS, and packed for another 3.5-cm with Luna 5 µm SCX 100 Å strong cation- exchange resin (Phenomenex, Torrance, CA) for on-line 2D-LC-MS/MS. The biphasic trapping column was connected to a 20-cm fused-silica analytical column (PicoTip Emitter, New Objective, 75 µm i.d.) with 3 µm C18 beads (ReproSil-Pur 120 C18-AQ, Dr. Maisch). For on- line 2D separation, ammonium acetate at concentrations of 0, 5, 10, 20, 50, 100, 200, 500 and 1000 mM were then sequentially injected using a 48-well autosampler from the sample vial to elute bound peptides from the precolumn to the analytical column with reversed-phase separation. The peptides were then separated with a 180-min linear gradient of 2-35% acetonitrile in 0.1% formic acid and at a flow rate of 250 nL/min. The LTQ-Orbitrap Velos was operated in a data-dependent scan mode. Full-scan mass spectra were acquired using the Orbitrap mass analyzer with a resolution of 60,000 with lock mass option enabled for the ion of m/z 445.120025.4 Up to 20 most abundant ions found in MS with charge state of 2 and above were sequentially isolated and sequenced in the linear ion-trap with a normalized collision energy of 35, an activation Q value of 0.25, and an activation time of 10 ms. Construction of MRM spectral library for the kinase peptides Skyline (version 3.5)5 was used to generate the spectral libraries for kinome peptides. All raw files obtained from discovery-mode proteomic analysis on the LTQ-Orbitrap Velos were searched by using MaxQuant (Version 1.3.0.5)6 against the human IPI protein database version 3.68 (87,062 entries). Initial precursor mass tolerance of 10 ppm and fragment mass deviation of 0.8 Th were set as the search criteria. The maximum number of miss-cleavages for trypsin was S3 set at two per peptide. Cysteine carbamidomethylation was considered as a fixed modification, whereas methionine oxidation and light desthiobiotinylation of lysine (+281.17394 Da, respectively) were included as variable modifications. The resulting MS/MS files were then imported into Skyline and filtered with a threshold score of 0.95. An interactive Skyline spectral library file containing tandem mass spectra of all desthiobiotin-C3-labeled kinase peptides was then generated. Retention time (RT) extraction and iRT calculation for kinase peptides To calculate the iRT score7 for each kinase peptide in the MRM kinome library, 10 peptides derived from the tryptic digestion mixture of bovine serum albumin (BSA) were selected to constitute the reference peptides for a new iRT scale. iRT values of these 10 standard peptides from BSA were calculated using their empirically measured RT from shotgun LC-MS/MS analysis by setting iRT scores of peptides AEFVEVTK and DAFLGSFLYEYSR as 0 and 100, respectively. The BSA peptide mixture was then added to desthiobiotin-C3-labeled kinase peptide mixture from the lysates of GM04429, GM00637, HEK293T, GM15876A, DU-145, HCT116, MCF7, U2OS and CEM cells and measured by 1D-LC-MS/MS on the LTQ Orbitrap Velos with a 130-min linear gradient. RTs were extracted for all BSA standard peptides as well as kinase peptides using the Skyline MS1 filtering workflow.8 The transformed iRT values for all the newly identified kinase peptides were calculated based on linear regression of iRT and experimentally measured RT of peptides with previously determined iRT score. With the accumulation of iRT scores for targeted kinase peptides, it is not necessary to include the BSA standard mixture for RT extraction and iRT calculation. Any common kinase peptides that yield a regression with a R2 value of 0.99 or higher were used as standard peptides to calculate the new iRT score for newly identified kinase peptides by Skyline. LC-MRM Analysis All LC-MRM experiments were carried out on a TSQ Vantage triple-quadrupole mass spectrometer equipped with a nanoelectrospray ionization source coupled to an EASY-nLC II system (Thermo Scientific). Samples were automatically loaded onto a 4-cm trapping column (150 µm i.d.) packed with 5 µm 120 Å reversed phase C18 material (ReproSil-Pur 120 C18-AQ, Dr. Maisch) at 3 µL/min. The trapping column was coupled to a 20-cm fused silica analytical column (PicoTip Emitter, New Objective, 75 µm i.d.) with 3 µm C18 beads (ReproSil-Pur 120 S4 C18-AQ, Dr. Maisch). The peptides were then separated with a 130-min linear gradient of 2- 35% acetonitrile in 0.1% formic acid and at a flow rate of 230 nL/min. The spray voltage was 1.9 kV. Q1 and Q3 resolutions were 0.7 Da and the cycle time was 5 s. To enable automated, multiplexed MRM analyses, we generated an interactive Skyline spectral library file containing tandem mass spectra of all unique desthiobiotin-C3 conjugated kinase peptides from 15 cell line samples and synthetic peptides, along with their iRT values,7 which were acquired from discovery-mode analysis on an LTQ Orbitrap Velos mass spectrometer, using Skyline (version 3.5).5 Collision energies were calculated using a linear equation that was specific to the TSQ Vantage instrument according to the Skyline default setting. Tryptic peptide mixture of BSA was analyzed in unscheduled MRM-mode prior to the analysis of the enriched desthiobiotin-C3 conjugated peptides. The linear predictor of empirical RT from iRT for targeted kinase peptides was then determined by the linear regression of RTs of BSA standard peptides obtained for the current chromatography setup. This linear predictor was rechecked between every five MRM sample analyses by injecting another BSA standard mixture. Three or four pairs of transitions were monitored for each light/heavy desthiobiotin-C3- labeled peptide in quantification experiments. These targeted transitions were monitored in five separate injections for each sample in scheduled MRM mode with a retention time window of 8 min.