KTH Uhlen Lab - Western blot

KTH Uhlen Lab - Western blot
Enhanced validation of antibodies for research applications

  • Organism: Homo sapiens
  • Instrument: Q Exactive HF
  • SpikeIn: Yes
  • Keywords: western blot, targeted proteomics, parallel reaction monitoring, prm, qprest, quantification
  • Lab head: Bjorn Forsstrom
Abstract
There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation “pillars” were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
Experiment Description
Liquid chromatography for targeted proteomics Liquid chromatography was preformed using as Ultimate 3000 binary RS nano system (Thermo Scientific) using an EASY-Spray ion source. All samples were stored in their lyophilized form and were resuspended by the autosampler prior to injection as 1 µg sample material was loaded onto an Acclaim PepMap 100 trap column (75 µm x 2 cm, C18, 3 µm, 100 Å), washed for 5 minutes at 0.250 µl/min with Solvent A (95 % H2O, 5 % DMSO, 0.1 % FA) and thereafter separated using a PepMap 800 C18 column (15 cm x 75 µm, 3 µm). The gradient went from Solvent A to Solvent B (90 % ACN, 5 % H2O, 5 % DMSO, 0.1 % FA) at a constant flow of 0.250 µl/min, up to 43 % Solvent B in 40 min, followed by an increase up to 55 % in 10 minutes followed by a steep increase to 100 % B in 2 min. Online LC-MS was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific). Spectral library generation for targeted proteomics An equimolar pool of QPrESTs was digested by trypsin according to the in-solution protocol described above. Only non-secreted protein targets were included from a set of QPrEST sequences previously generated based on being frequently identified in shotgun proteomics experiments performed on cell lines. The recombinant protein sequences had to generate at least one proteotypic peptide upon trypsin digestion. For initial screening, 50 fmol of each QPrEST was loaded onto column and a Top5 MS-method was performed with master scans at 60,000 resolution (mass range 300 1600 m/z, AGC 3e6), followed by five consecutive MS/MS scans at 30,000 resolution (AGC 1e5, underfill ratio 0.1 %) with normalized collision energy set to 25. Raw mass spectrometry files were processed using MaxQuant, using the search engine Andromeda and searched against a FASTA file containing QPrEST sequences spiked into the E. coli proteome (BL21, Uniprot-ID: #UP000002032), which was used for recombinant protein production Identified peptides were further processed by only allowing proteotypic peptides mapping to one single human gene (defined by Swiss-Prot v72), thereby excluding peptides with potential misscleavages and peptides including any methionine. Data independent MS acquisition targeted proteomics Full MS master scans at 60,000 resolution (mass range 300-1600 m/z, AGC 1e6) were followed by 20 data-independent acquisitions MS/MS at 60,000 resolution (AGC 1e6) defined by a scheduled Parallel Reaction Monitoring (PRM) method. Precursors were isolated in a scheduled sequence with a 1.2 m/z isolation window and the maximum injection time was set to 105 ms for both MS and MS/MS resulting in a duty cycle of 2.7 s. The isolation list was split into two consecutive LC-runs, targeting 120 paired light and heavy peptides per injection.
Sample Description
Cell line cultivation used for targeted proteomics Eight different cell-lines (A431, Hep G2, A549, HeLa, HEK 293, U-2 OS, RT4, and SH-SY5Y) were cultivated at 37 °C in a humidified atmosphere containing 5 % CO2. A549 and SH-SY5Y were cultivated in Dulbecco’s modified Eagle’s Medium (Sigma Aldrich). HEK 293, HeLa and Hep G2 were cultivated in Minimum Essential Medium Eagle (Sigma Aldrich). U-2 OS and RT4 were cultivated in McCoy’s medium (Sigma Aldrich) and A431 was cultivated in RPMI-1640 (Sigma Aldrich). All media were supplemented with 10 % Fetal Bovine Serum (Sigma Aldrich). Media for HEK 293, HeLa and Hep G2 were supplemented with 1 % MEM non-essential Amino Acid Solution (Sigma Aldrich) and media for and Hep G2 were also supplemented with 1 % L Glutamine (Sigma Aldrich). The cells were cultivated up to 80 % confluence and cell numbers were counted with a Scepter 2.0 Cell Counter (Merck Millipore, Billerica, MA, USA) before pellets were collected and stored at -80 °C. Cell lysis for targeted proteomics Cells were dissolved in Lysis buffer (100 mM Tris-HCl, 4 % SDS, 10 mM DTT, pH 7.6) and incubated at 95 °C in a thermomixer for 5 minutes at 600 rpm and thereafter sonicated at 50 % amp (1 s pulse, 1 s hold) for one minute. The same cell lysate was subjected for mass spectrometry analysis and western blot analysis. Filter aided sample preparation One QPrEST mastermix was prepared to represent endogenous levels of U 2 OS with light to heavy peptide ratios as close to one as possible. The same amount of mastermix was spiked into cell lysates consisting of one million cells. The lysate was diluted with denaturing buffer (8 M Urea, 100 mM Tris-HCl, pH 8.5) and centrifuged through a 0.22 µm spin filter (Corning, Corning, NY, USA). Trypsin digestion was performed using a previously described filter-aided sample preparation (FASP) method23. Trypsin was added in a 1:50 enzyme to substrate ratio and incubated overnight at 37°C. Peptides were extracted by solid-phase extraction using in-house prepared C18 StageTips following the protocol described above. Desalted peptides were vacuum dried before LC-MS analysis.
Created on 8/22/18, 8:28 AM
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
LINCS_P100_DIA_Plate82_annotated_minimized_2019-06-13_13-17-49.sky.zip (110 MB)2019-06-1390961922,76491
LINCS_P100_DIA_Plate80_annotated_minimized_2019-06-13_12-13-28.sky.zip (121 MB)2019-06-1390961922,76496
LINCS_GCP_Plate71b_annotated_minimized_2019-06-10_18-12-11.sky.zip (12 MB)2019-06-10818116283018
LINCS_GCP_Plate71a_annotated_minimized_2019-06-10_18-10-31.sky.zip (5 MB)2019-06-1081811628548
LINCS_P100_DIA_Plate77_annotated_minimized_2019-06-10_16-17-36.sky.zip (81 MB)2019-06-1090961922,76266
LINCS_P100_DIA_Plate70_annotated_minimized_2019-06-03_13-47-33.sky.zip (140 MB)2019-06-0890961922,76496
LINCS_P100_DIA_Plate82_annotated_minimized_2019-06-03_16-03-24.sky.zip (110 MB)2019-06-0890961922,76491
LINCS_P100_DIA_Plate76_annotated_minimized_2019-06-07_15-29-16.sky.zip (100 MB)2019-06-0790961922,75866
LINCS_P100_QC_Plate80_annotated_minimized_2019-06-04_16-27-57.sky.zip (7 MB)2019-06-04882417496
LINCS_P100_QC_Plate82_annotated_minimized_2019-06-04_15-46-59.sky.zip (8 MB)2019-06-04882418396
LINCS_P100_QC_Plate78_annotated_minimized_2019-06-03_15-57-25.sky.zip (7 MB)2019-06-03882316196
LINCS_P100_DIA_Plate70_annotated_minimized_2019-06-03_13-47-33.sky.zip (140 MB)2019-06-0390961922,76496
LINCS_P100_QC_Plate70_annotated_minimized_2019-06-03_13-48-35.sky.zip (7 MB)2019-06-03882416596
all combined_2019-03-21_20-40-14.sky.zip (46 MB)2019-05-31133575268,5337
20180518 multiplex quantification - APOA4 only samples_2018-05-18_12-13-20.sky.zip (265 MB)2019-05-311364864
20180518 multiplex quantification only samples_2018-05-18_11-58-07.sky.zip (380 MB)2019-05-3128357050268
20180509_prest-kinetics_2018-05-09_18-52-06.sky.zip (120 MB)2019-05-311613628684820
500 µE (PG-500uE_2ul_2019-05-16_15-23-09).sky.zip (7 MB)2019-05-292710510555811
Huidan-LHCSR3-Trypsin-PRM_FINAL2_2019-05-16_17-33-35.sky.zip (3 MB)2019-05-2920611225758
Huidan-LHCSR3-LysC-selected-tSIM-PRM_FINAL_2019-05-16_17-31-57.sky.zip (13 MB)2019-05-2931681366528
200µE (R5-8_V2_2019-05-16_15-31-29).sky.zip (8 MB)2019-05-2928979751616
matchedmatrixcalcurve_yeasthela_2019-05-28_13-23-23.sky.zip (1 GB)2019-05-283,22827,28927,28985,17542
matchedmatrixcalcurve_yeastwater_2019-05-24_12-06-14.sky.zip (1 GB)2019-05-243,73136,39336,39395,63942
20150514_Plasma_remove_points_2019-05-22_18-51-25.sky.zip (162 MB)2019-05-2220989862412
matchedmatrixcalcurve_csf_2019-05-22_14-34-12.sky.zip (531 MB)2019-05-224,00716,22916,22947,85630
matchedmatrixcalcurve_yeastyeast_2019-05-22_12-49-50.sky.zip (1 GB)2019-05-223,48230,60230,60290,48842
Janschitz et al_PRM_results_Panorama upload_2019-05-06_18-03-18.sky.zip (611 MB)2019-05-094475833712
LINCS_GCP_Plate76_annotated_minimized_2019-04-29_15-46-06.sky.zip (16 MB)2019-04-29818116287833
LINCS_P100_DIA_Plate51_annotated_minimized_2017-08-02_16-21-22.sky.zip (43 MB)2019-04-26909619292496
LINCS_P100_DIA_Plate41_annotated_minimized_2017-02-16_14-31-38.sky.zip (128 MB)2019-04-2690961921,00296
LINCS_P100_DIA_PlateAD2_annotated_minimized_2019-04-18_11-17-21.sky.zip (52 MB)2019-04-18909619278892
LINCS_P100_QC_PlateAD2_annotated_minimized_2019-04-18_11-15-52.sky.zip (6 MB)2019-04-18882413292
LINCS_GCP_Plate77_annotated_minimized_2019-04-17_09-22-00.sky.zip (19 MB)2019-04-17818116291233
LINCS_GCP_Plate75_annotated_minimized_2019-04-16_13-40-09.sky.zip (26 MB)2019-04-16818116291233
LINCS_GCP_Plate74_annotated_minimized_2019-04-16_13-21-11.sky.zip (24 MB)2019-04-16808016080533
LINCS_GCP_Plate73_annotated_minimized_2019-04-16_11-45-08.sky.zip (24 MB)2019-04-16818116289433
LINCS_P100_QC_Plate75_annotated_minimized_2019-04-08_16-33-18.sky.zip (4 MB)2019-04-08882415666
LINCS_P100_DIA_Plate75_annotated_minimized_2019-04-08_16-26-05.sky.zip (72 MB)2019-04-0890961922,76466
LINCS_P100_QC_Plate76_annotated_minimized_2019-04-08_15-34-30.sky.zip (5 MB)2019-04-08882415066
Dee Dee Luu et. al_The immunogenic microbial peptide RaxX represents an unclassified group of sulfated ribosomally synthesized and post-translationally modified peptides_2019-04-02_12-44-12.sky.zip (1 MB)2019-04-062221318
LINCS_P100_DIA_Plate73_annotated_minimized_2019-04-02_12-31-00.sky.zip (75 MB)2019-04-0290961922,76466
LINCS_P100_QC_Plate74a_annotated_minimized_2019-04-01_16-25-26.sky.zip (2 MB)2019-04-01882417436
LINCS_P100_DIA_Plate74a_annotated_minimized_2019-04-01_16-19-30.sky.zip (39 MB)2019-04-0190961922,76236
LINCS_P100_QC_Plate73_annotated_minimized_2019-03-28_10-49-18.sky.zip (5 MB)2019-03-28882416266
LINCS_P100_QC_Plate74_annotated_minimized_2019-03-27_16-36-40.sky.zip (6 MB)2019-03-27882417182
LINCS_P100_DIA_Plate74_annotated_minimized_2019-03-27_16-18-55.sky.zip (98 MB)2019-03-2790961922,76284
LINCS_P100_QC_Plate70_annotated_minimized_2019-03-13_14-12-46.sky.zip (7 MB)2019-03-13882416596
HLF with ladder_GUnames_2018-08-15_19-59-17.sky.zip (176 KB)2019-03-086045451
U87MG with ladder_GUnames_2018-08-15_20-02-38.sky.zip (429 KB)2019-03-08901181181
IgA with ladder_GUnames_2018-08-15_20-00-17.sky.zip (358 KB)2019-03-08801251251
HNE with ladder_GUnames_2018-08-15_19-59-46.sky.zip (174 KB)2019-03-086038381
PGM OG with ladder_GUnames_2018-08-15_20-01-34.sky.zip (370 KB)2019-03-08201061061
IgG with ladder_GUnames_2018-08-15_20-01-05.sky.zip (228 KB)2019-03-087061611
Fet NG with ladder_GUnames_2018-08-15_19-58-42.sky.zip (229 KB)2019-03-087063631
CBHI OG with ladder_GUnames_2018-08-15_19-58-20.sky.zip (93 KB)2019-03-082020201
CBHI NG with ladder_GUnames_2018-08-15_19-57-24.sky.zip (109 KB)2019-03-083028281
BLF with ladder_GUnames_2018-08-15_19-55-59.sky.zip (302 KB)2019-03-088085851
Fig3_U87MG_iRT_ladder_2018-08-15_19-50-07.sky.zip (429 KB)2019-03-08901191191
Fig5_SupFig3_Dextran_Spectral_Library_2018-08-15_19-53-02.sky.zip (40 KB)2019-03-08103181
Fig1_SupFig1_Ladder_tech_reps_2018-08-15_19-48-46.sky.zip (797 KB)2019-03-0810151510
Fig6_2019-01-26_23-48-44.sky.zip (63 MB)2019-03-088011634212
Fig2_SupFig2_iRT_linear.sky.zip (6 MB)2019-03-0820183216
PRM-Identifying the N-terminal amino acid for a TAG start codon_2019-03-04.sky.zip (3 MB)2019-03-05266583
PRM-Identifying the N-terminal amino acid for an ATG start codon_2019-03-04.sky.zip (1 MB)2019-03-05266563
Cortex_50_targets_PRM_finaL_20181212_2019-01-15_15-55-34.sky.zip (5 MB)2019-03-01551332582,2633
Pancreas_50_targets_PRM_final_20181210_2019-01-15_15-52-49.sky.zip (5 MB)2019-03-01631342602,0613
sasp_panel_ras_2019-02-26_13-24-23.sky.zip (51 MB)2019-02-2674730,02544,411576,8660
sasp_panel_irradiation_2019-02-26_13-00-53.sky.zip (17 MB)2019-02-261729,62414,598189,6470
sasp_panel_atazanavir_2019-02-26_11-45-06.sky.zip (27 MB)2019-02-2637615,60123,172301,0320
sasp_panel_all_inducers_2019-02-26_11-18-00.sky.zip (17 MB)2019-02-261729,62414,598189,6470
QEPlus_2019-02-05_05-55-27.sky.zip (23 MB)2019-02-0419192414469
QEPlus_2019-02-03_11-26-48.sky.zip (23 MB)2019-02-0319192414469
Gigas-6-10-DIA.sky.zip (1 GB)2019-01-309,04732,31233,292348,20120
2017-09-06-GigasDilutionCurve.sky.zip (118 MB)2019-01-301851511728
2017-09-06-Gigas-SRM-ReplicatesOnly-PostDilutionCurve-emma.sky.zip (244 MB)2019-01-3017474713795
WB_PK_bi_scFv_2019-01-18_23-49-08.sky.zip (2 MB)2019-01-2215107062
Plasma_PK_bi_scFv_2019-01-18_23-51-46.sky.zip (2 MB)2019-01-2215107058
Plasma_PK_bi_scFv_2019-01-18_23-51-46.sky.zip (2 MB)2019-01-1815107058
WB_PK_bi_scFv_2019-01-18_23-49-08.sky.zip (2 MB)2019-01-1815107062
20190110 - Ajinomoto Complete_2019-01-10_13-31-48.sky.zip (23 MB)2019-01-1163434161238
2017-geoduck-SRM.sky.zip (191 MB)2019-01-09144949146124
2017-06-02_Geoduck-DIA-2016.sky.zip (1 GB)2019-01-098,07730,35230,659298,34518
Thomas C.glu isopentenol production targeted proteins_2019-01-07_16-39-19.sky.zip (6 MB)2019-01-089414115648
TGC SRM Reference.sky.zip (7 MB)2019-01-072985295291,5875
TGC SRM Tissue.sky.zip (36 MB)2019-01-072213313311,19015
180913_OBAL_picked-rep1_2018-12-20_10-43-34.sky.zip (10 MB)2018-12-2015224420638
160616_OBAL_95_samples_quant_high_quality_2018-12-20_10-50-47.sky.zip (77 MB)2018-12-2013173415895
160616_OBAL_95_samples_quant_2018-12-20_10-50-06.sky.zip (77 MB)2018-12-2015224420695
170208_OBAL_Samples_refined_2018-12-20_10-37-30.sky.zip (39 MB)2018-12-2015224421096
170705_HER2_positive_ccr_2018-12-11_11-18-06.sky.zip (5 MB)2018-12-13212126010
171128_Cal_Ext_Rev_BG_Ecoli_Exp4_Rep5_2018-12-11_11-24-53.sky.zip (7 MB)2018-12-132242018
180322_HER2_peptide-Ecoli-Exp6_Rep3_2018-12-11_11-22-26.sky.zip (3 MB)2018-12-1321212609
170705_HER2_negative_ccr_2018-12-11_11-21-52.sky.zip (12 MB)2018-12-13318189025
ADH_DIA_092018_2018-12-12_14-11-02.sky.zip (293 MB)2018-12-1211112108207
Calibration A1AG1+2 and IGHA1_2018-11-02_12-10-57.sky.zip (8 MB)2018-12-058142713024
Protein quantification in DBS from 15 volunteers SDC extraction_2018-11-02_10-52-13.sky.zip (3 MB)2018-12-058142713015
Comparison of 3mm DBS punch and 3ul liquid blood_2018-11-02_10-48-05.sky.zip (3 MB)2018-12-058142713012
Calibration in SDC_2018-11-02_12-16-42.sky.zip (18 MB)2018-12-058142713071
Calibration SAA4_2018-11-02_12-15-37.sky.zip (16 MB)2018-12-058142713045
Calibration SAA1+2_2018-11-02_12-14-22.sky.zip (17 MB)2018-12-058142713048