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.

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.

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.