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Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

2015; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês

10.1074/mcp.o115.054940

1535-9484

Jacob J. Kennedy, Ping Yan, Lei Zhao, Richard G. Ivey, Uliana J. Voytovich, Heather D. Moore, Chenwei Lin, Era L. Pogosova‐Agadjanyan, Derek L. Stirewalt, Kerryn W. Reding, Jeffrey R. Whiteaker, Amanda G. Paulovich,

bioluminescence and chemiluminescence research

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. Cell signaling research is faced with the challenging task of interrogating increasingly large numbers of analytes in "systems biology" approaches, while maintaining the high standards of integrity and reproducibility traditionally associated with the scientific approach. For example, studies interrogating complex systems, such as protein signaling networks, require quantification technologies capable of sensitive, specific, multiplexable, and reproducible application. However, recent reports have highlighted alarmingly high rates of irreproducibility in fundamental biological and pre-clinical studies (1..C. G. Begley, L. M. Ellis, Drug development: Raise standards for preclinical cancer research. Nature. 483, 531–533,Google Scholar, 2.Prinz F. Schlange T. Asadullah K. Believe it or not: how much can we rely on published data on potential drug targets?.Nat. Rev. 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To improve the translation of cell signaling discoveries into clinical application, we need reproducible and transferable technologies that enable higher throughput quantification of protein phosphorylation. Signaling dynamics through post-translational modifications (e.g. phosphorylation) are predominantly measured by Western blotting. Although this technique has led to many discoveries and is the de facto "gold standard," it suffers from many drawbacks. Western blotting is a low throughput approach applied to individual analytes (i.e. no multiplexing) and is susceptible to erroneous interpretation when applied quantitatively (6.Janes K.A. An analysis of critical factors for quantitative immunoblotting.Sci. Signal. 2015; 8: rs2Crossref PubMed Scopus (138) Google Scholar). Alternative immunoassay platforms have emerged (e.g. immunohistochemistry, ELISA, mass cytometry, and bead-based or planar arrays), but suffer from similar limitations, namely specificity issues (because of cross-reactivity of antibodies), poor standardization, and difficulties in multiplexing. One alternative for quantifying phosphorylation is targeted, multiple reaction monitoring (MRM) 1The abbreviations used are:MRMmultiple reaction monitoringIMACimmobilized metal affinity chromatographySOPstandard operating protocolsDDRDNA damage responsePBMCperipheral blood mononuclear cellIRionizing radiationMMSmethyl methanesulfonateLODlimit of detectionLLOQlower limit of quantificationWBWestern blotCPTACClinical Proteomic Tumor Analysis Consortium. MS, a widely deployed technique in clinical laboratories for quantification of small molecules (7.Chace D.H. Kalas T.A. A biochemical perspective on the use of tandem mass spectrometry for newborn screening and clinical testing.Clin. 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PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.Mol. Cell. Proteomics. 2012; 11: 187-201Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) have been implemented with shotgun mass spectrometry. Although anti-peptide antibodies can also be used to enrich individual phosphopeptides upstream of MRM (28.Whiteaker J.R. Zhao L. Yan P. Ivey R.G. Voytovich U.J. Moore H.D. Lin C. Paulovich A.G. Peptide immunoaffinity enrichment and targeted mass spectrometry enables multiplex, quantitative pharmacodynamic studies of phospho-signaling.Mol. Cell. Proteomics. 2015; (pii: mcp.O115.054940. [Epub ahead of print])Abstract Full Text Full Text PDF Scopus (52) Google Scholar), the generation of these reagents is time-consuming and costly, limiting widespread uptake. multiple reaction monitoring immobilized metal affinity chromatography standard operating protocols DNA damage response peripheral blood mononuclear cell ionizing radiation methyl methanesulfonate limit of detection lower limit of quantification Western blot Clinical Proteomic Tumor Analysis Consortium. Phosphopeptide enrichment based on metal affinity chromatography has recently matured into a reproducible approach (29.Bodenmiller B. Mueller L.N. Mueller M. Domon B. Aebersold R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome.Nat. Methods. 2007; 4: 231-237Crossref PubMed Scopus (506) Google Scholar). Immobilized metal affinity chromatography (IMAC) is widely used in discovery phosphoproteomic studies to enrich phosphopeptides upstream of shotgun-based mass spectrometry (30.Muszyńska G. Dobrowolska G. Medin A. Ekman P. Porath J.O. Model studies on iron(III) ion affinity chromatography. II. Interaction of immobilized iron(III) ions with phosphorylated amino acids, peptides and proteins.J. Chromatogr. 1992; 604: 19-28Crossref PubMed Scopus (101) Google Scholar, 31.Kanshin E. Michnick S. Thibault P. Sample preparation and analytical strategies for large-scale phosphoproteomics experiments.Semin. Cell Dev. Biol. 2012; 23: 843-853Crossref PubMed Scopus (34) Google Scholar). We hypothesized that a subset of the cellular phosphoproteome with favorable binding characteristics to the IMAC resin might be reproducibly recovered for quantification when coupled with quantitative MRM mass spectrometry, enabling robust IMAC-MRM assays without the need for an antibody. In this report, we: (1) demonstrate the feasibility of generating analytically robust, multiplex IMAC-MRM assays for quantifying cellular phospho-signaling, (2) present a semi-automated, 96-well format magnetic bead-based protocol for IMAC enrichment, (3) provide a catalogue of phosphopeptides that are highly amenable to IMAC-MRM quantification, and (4) make publicly available standard operating protocols (SOP) and fit-for-purpose analytical validation data for IMAC-MRM assays targeting 107 phospho-analytes, providing a community resource for study of the DNA damage response. The data suggest that the IMAC-MRM approach is generally applicable to signaling pathways, enabling wider interrogation of signaling networks. All LC-MS/MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (32.Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Perez-Riverol Y. Reisinger F. Rios D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-9Crossref PubMed Scopus (1596) Google Scholar) with the data set identifier PXD002363. The MS/MS spectra and MRM transitions to these 190 phosphopeptide targets can be found in supplemental data File S1.zip. All MRM data (response curves and cell line measurements) are available in the supplemental File S2–S5.zip. Characterization data, SOPs, and assay information are available on the CPTAC Assay Portal (www.assays.cancer.gov). Urea (#U0631), Trizma base (#T2694), iodoacetamide (IAM, #A3221), iron (III) chloride (FeCl3, #157740), ammonia (28–30% ammonium hydroxide, #320145), and ammonium bicarbonate (NH4HCO3, #A6141) were obtained from Sigma (St. Louis, MO). Acetonitrile (MeCN, #A955), water (H2O, #W6, LCMS Optima® grade), tris(2-carboxyethyl)phosphine (TCEP, #77720), trifluoroacetic acid (TFA, #28901), and phosphate buffered saline (PBS, #BP-399–20) were obtained from Thermo Fisher Scientific (Waltham, MA). Formic acid (FA, #1.11670.1000) was obtained from EMD Millipore (Billerica, MA). Sequencing grade trypsin (#V5113) was obtained from Promega (Madison, WI). Purified and crude heavy stable isotope-labeled peptides were obtained from New England Peptide (Gardner, MA) and ThermoFisher Scientific. Purified synthetic peptides were >95% pure as measured by HPLC. Peptide stock concentrations were determined by amino acid analysis (AAA) at New England Peptide. Peptide quality control included HPLC chromatograms, MALDI mass spectrum confirming the molecular weight, and AAA analysis to confirm correct amino acid stoichiometries (provided by vendor). Aliquots were stored in 3% MeCN/0.1% FA at −80 °C until use. Crude peptides were synthesized on a 1.0 μmol scale, purified by solid phase extraction to remove all nonpeptide contaminants (average purity ∼75%), and the correct molecular mass verified by MS. Aliquots were shipped lyophilized and resuspended in 1 ml 3% MeCN/0.1% FA or were shipped in 0.4 ml 50% MeCN/0.1% TFA. Aliquots were stored at −80 °C until use. All peptide sequences were synthesized with free N-terminal and C-terminal amino acids. S-carbamidomethylated versions of cysteine residues (CAM-C) were used. The C-terminal arginine or lysine was uniformly labeled with [13C and 15N] labeled atoms. The human mammary epithelial cell line MCF10A was obtained from the ATCC (Manassas, VA) and grown at 37 °C and 5% CO2 in DMEM/F12 1:1 (Invitrogen #11320) supplemented with 5% horse serum (Invitrogen), 10 mg/ml of insulin (Sigma #I6634), 20 ng/ml of EGF (PeproTech #AF-100–15), 0.5 mg/ml of hydrocortisone (Sigma #H-0888), 100 ng/ml of cholera toxin (Sigma #C-8052), 100 units/ml of penicillin, and 100 mg/ml streptomycin. Cells that were heavy-labeled for the discovery experiment were grown in the presence of 0.1 mg/ml l-Arginine (13C6, 15N4,) and 0.1 mg/ml l-Lysine (13C6, 15N2) (Cambridge Isotope Laboratories, Tewksbury, MA, # CNLM-539-H-0.1 and CNLM-291-H-0.1) in the medium. Cells were grown to 80% confluence in 100 mm plates before treatment with methyl methanesulfonate (MMS, Sigma #129925) or ionizing radiation (see below). After incubation for the indicated times, growth medium was removed, cells rinsed in 0.25% trypsin/EDTA solution (Gibco #25200–056), and lifted off the plates by incubation in a fresh aliquot of 0.25% trypsin/EDTA solution at 37 °C, 5% CO2. When cells had lifted from the plate, the trypsin was quenched by the addition of three volumes of DMEM/F12 with 5% horse serum. Following informed consent, peripheral blood mononuclear cells (PBMC) were obtained from healthy adults (Fred Hutchinson Cancer Research Center IRB #9026, 8233, and 6421). Whole blood samples were collected by venipuncture into K2EDTA tubes and maintained at ambient temperature during transportation to the laboratory. PBMC were isolated from whole blood using Histopaque-1077 (Sigma #10771) density gradient. Whole blood was diluted with an equal volume of PBS, layered over a one-third volume of Histopaque (density = 1.077g/ml) at room temperature, and centrifuged at 400 × g for 30 min with no brake. PBMCs were harvested from the Histopaque-plasma interface, washed twice in PBS, and treated once with Red Blood Cell Lysis Buffer (5-Prime #2301310). The T-cell population was activated and expanded using a 1:1 cell to bead ratio of CD3/CD28 Dynabeads (Invitrogen # 111–31D). Culturing was done in Advanced RPMI 1640 (Gibco #12633012) supplemented with 10% heat-inactivated FBS (Hyclone #SH30071.03HI), 100 units/ml of penicillin, 100 units/ml streptomycin (Gibco #15070–063), 2 mm l-Glutamine (Gibco #25030–081), 100 units/ml IL2 (Life Technologies #PHC0027). Cells were cultured for 9 days at 37 °C and 5% CO2 with fresh growth medium added every two to 3 days. On day 7, cells were resuspended in fresh medium at 1.25 × 106 cells/ml in T75 flasks and allowed to equilibrate at 37 °C, 5% CO2 for 36 to 40 h before treatment with DNA-damaging agents. Cells treated with 10 Gy ionizing radiation (IR) were irradiated in a JL Shepherd Mark I irradiator using a 137Cs source delivering a dose rate of 4.7 Gy/minute. All flasks were returned to the incubator for the indicated length of time at 37 °C, 5% CO2, and then cells were harvested and lysates generated. Control cells were mock-irradiated; the mock-irradiated cells were handled in precisely the same manner as the irradiated cells but the irradiator was not turned on. MMS was diluted in growth medium just prior to addition to cell cultures for a final concentration of 0.5 mm. Cells were incubated for the indicated length of time at 37 °C, 5% CO2 before being harvested. Cells were harvested in prechilled tubes, aliquots were removed for counting, and cells were spun down and washed 2 times in an equal volume of ice cold PBS. Cell count was determined with a Beckman Coulter Z1 Particle Counter. Cells were lysed at 5 × 107 cells/ml in freshly prepared ice cold Urea Lysis Buffer (6 m Urea, 25 mm Tris (pH8.0), 1 mm EDTA, 1 mm EGTA containing protease and phosphatase inhibitors (Sigma, #P0044, #P5726, and #P8340)). Lysates were sonicated 2 × 12 s and then cleared by centrifugation at 20,000 × g, 10 min at 4 °C. Supernatants were transferred to cryo-vials, stored in liquid nitrogen and thawed on ice. Protein lysate concentration was measured in triplicate using Micro BCA Protein Assay Kit (Thermo # 23235). Prior to downstream analysis, all samples were blinded and the analysis order randomized within and across batches. Lysates were reduced in 6% TCEP for 30 min at 37 °C with shaking, followed by alkylation with 50 mm IAM in the dark at room temperature. Lysates were then diluted 1:10 with 200 mm TRIS, pH 8, before trypsin was added at a 1:50 trypsin/protein ratio by mass. After 2 h, a second trypsin aliquot was added at a 1:100 trypsin/protein ratio. Digestion was carried out overnight at 37 °C with shaking. After 16 h, the reaction was quenched with FA (final concentration 1% by volume). For MRM studies, a mix of SIS peptides was spiked at 50 fmol (250 fmol/mg protein), levels high enough above the LLOQ so as not to contribute unnecessarily to the assay CV and close to expected endogenous levels so that the peak area ratio was not outside of the range of 100:1 and 1:100. The mixture was desalted using Oasis HLB 96-well plates (Waters #WAT058951) and a positive pressure manifold (Waters #186005521). The plate wells were washed with 3 × 400 μl of 50% MeCN/0.1% FA, and then equilibrated with 4 × 400 μl of 0.1% FA. The digests were applied to the wells, then washed with 4 × 400 μl 0.1% FA before being eluted drop by drop with 3 × 400 μl of 50% MeCN/0.1% FA. The eluates were lyophilized, followed by storage at −80 °C until use. For LC-MRM analysis, IMAC enrichment was performed according to Ficarro et al. (33.Ficarro S.B. Adelmant G. Tomar M.N. Zhang Y. Cheng V.J. Marto J.A. Magnetic Bead Processor for Rapid Evaluation and Optimization of Parameters for Phosphopeptide Enrichment.Anal. Chem. 2009; 81: 4566-4575Crossref PubMed Scopus (123) Google Scholar) with the following modifications. Ni-NTA-magnetic beads (Qiagen, Valencia, CA) were stripped with 100 mm EDTA and then incubated in a 10 mm FeCl3 solution to prepare magnetic Fe3+-NTA-agarose beads. Peptide enrichment was performed out of 200 μg of lysate digest reconstituted in 200 μl of 0.1% TFA in 80% ACN in 96-well plates (Thermo Kingfisher #97002540) loaded with 100 μl magnetic beads from 5% bead suspension. The plate containing samples and beads was mixed on the titer plate shaker (Lab Line Instruments) at speed 4 for 30 min at room temperature. A KingFisher magnetic particle processor (Thermo Fisher) with a PCR head was used for all bead handling. Beads were mixed in the incubation plate for 5 min, and then transferred for three washes (1 min each in 0.1% TFA in 80% ACN, 200 μl). Enriched peptides were eluted in 200 μl of 1:1 acetonitrile/1:20 ammonia:water for 5 min. The elution plate was dried using a GenVac vacuum centrifugation system and samples were covered with adhesive foil and frozen at −80 °C until analysis. Fe3+-NTA magnetic agarose beads were recycled by washing used beads 3× with 1 ml water and then following the procedure for preparing Ni-NTA-magnetic beads described above. Targeted LC-MRM-MS analysis was performed by a trap-elute configuration on a nanoLC- 2D and cHiPLC-Nanoflex system (Eksigent Technologies, Dublin, CA) coupled to a 5500 QTRAP mass spectrometer (ABSciex, Foster City, CA) by an Advance CaptiveSpray source (Michrom Bioresources, Auburn, CA). Mobile phases consisted of 0.1% FA in water (A) and 90% MeCN with 0.1% FA (B). 4 μl of sample was loaded onto a 200 μm x 0.5 mm ChromXP C18-CL 3 μm 120 Å column (Eksigent) using the following method: hold at 1% B and 10 μl/min for 2 min, gradient from 1 to 50% B and 2 μl/min for 0.1 min, hold 50% B and 2 μl/min for 62.9 min, gradient from 50 to 1% B and 10 μl/min for 0.1 min, re-equilibrate at 1% B and 10 μl/min for 14.9 min. The column temperature was 40 °C. The sample was injected onto the analytical column at 2 min and separated by a 75 μm x 15 cm ChromXP C18-CL 3 μm 120 Å column (Eksigent) using the following gradient method: hold at 1% B for 3 min, gradient from 1 to 10% B for 7 min, gradient from 10 to 25% B for 30 min, gradient from 25 to 40% B for 15 min, gradient from 40 to 60% B for 10 min, gradient from 60 to 90% B for 1 min, hold 90% B for 3 min, gradient from 90 to 1% B for 1 min, re-equilibrate at 1% B for 10 min. The flow rate was 300 nL/min and the column temperature was 40 °C. The MS was used in positive ion mode with parameters consisting of a 1200 V ion spray voltage, curtain gas setting of 10, nebulizer gas setting of 0, and an interface heater temperature of 110 °C. CE was described above, DP was set to 90, EP was set to 10, CXP was set to 10, and Q1 and Q3 set to unit/unit resolution (0.7 Da).Throughout the method, the actual cycle time remained at or below 2 s allowing for measurement of at least 10 points across the peaks. MRM peak integration was performed by Skyline (34.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinforma. Oxf. Engl. 2010; 26: 966-968Crossref PubMed Scopus (2988) Google Scholar), and the integrations were manually inspected to ensure correct peak detection, absence of interferences, and accurate integration. Reported peak areas are the sum of the peak area and background area reported by Skyline. Peak specificity between the light (or endogenous) and heavy (or SIS) MRM signal was defined as the detection of ≥1 transition from the endogenous peptide exactly co-eluting with ≥2 transitions from the stable isotope-labeled peptide, with the relative intensity of the light transition(s) deviating no more than 20% compared with the relative intensity of the corresponding heavy transitions. Integration results were exported to the program R for linear regression and statistical analysis. Peptide concentrations are calculated as the peak area ratio of the quantifying transition times the concentration of SIS peptide. Spectral libraries created in Skyline from discovery proteomics data and synthetic peptide QC data were used to select transitions for optimization. The MS/MS spectra to the phosphopeptide targets can be found in supplemental data File S1. Optimal collision energy for a hybrid triple quadrupole/linear ion trap mass spectrometer (5500 QTRAP) for a subset of the peptides was determined by injecting 50 fmol standard peptide solutions into a flow of 30% MeCN, 0.1% FA at a flow rate of 1 μl/min. Optimal values were determined by ramping the potentials. From these results,