Mass spectrometry–based quantitative proteomic analysis has proven valuable for clinical and biotechnology related research and development. Driving this value have been improvements in the sensitivity, resolution, and robustness of mass analyzers. However, manual sample preparation protocols are often a bottleneck for sample throughput and can lead to poor reproducibility, especially for applications where thousands of samples per month must be analyzed. To alleviate these issues, we developed a ‘cells-to-peptides’ automated workflow for Gram-negative bacteria and fungi that includes cell lysis, protein precipitation, resuspension, quantification, normalization, and tryptic digestion. The workflow takes two hours to process 96 samples from cell pellets to the initiation of the tryptic digestion step and can process 384 samples in parallel. We measured the efficiency of protein extraction from various amounts of cell biomass and optimized the process for standard liquid chromatography-mass spectrometry systems. The automated workflow was tested by preparing 96 E. coli samples and quantifying over 600 peptides that resulted in a median coefficient of variation of 15.8%. Similar technical variance was observed for three other organisms as measured by highly-multiplexed LC-SRM-MS acquisition methods. These results show that this automated sample preparation workflow provides robust, reproducible proteomic samples for high-throughput applications.

Targeted SRM methods were developed with the assistance of in-house built spectral libraries of the microbes. SRM selection criteria excluded peptides with Met/Cys residues, tryptic peptides followed by additional cut sites (KK/RR), and peptides with proline adjacent to K/R cut sites. All possible doubly charged peptides were screened for y-series ions to establish the peptide identity and the most sensitive transitions. The SRM targeted proteomic assays were performed on an Agilent 6460QQQ mass spectrometer system coupled with an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, CA). Mobile phase A consisted of 0.1% FA (Thermo Scientific) in LC-MS grade water (Burdick & Jackson, Muskegon, MI), and mobile phase B consisted of 0.1% FA in LC-MS grade acetonitrile (Burdick & Jackson). Twenty (20) µg peptides were separated on an Ascentis Express Peptide C18 column [2.7-mm particle size, 160-Å pore size, 5-cm length × 2.1-mm inside diameter (ID), coupled to a 5-mm × 2.1-mm ID guard column with the same particle and pore size, operating at 60°C; Sigma-Aldrich, St. Louis, MO] operating at a flow rate of 0.4 ml/min via gradients depending on the purpose of our investigations. To comprehensively evaluate the variance of automated sample preparation platform for microorganism we investigated, highly multiplexed assays that target hundreds of peptides were developed in either a single, scheduled 25 minute-gradient UHPLC-SRM-MS analysis for E. coli or a single, scheduled 30 minute-gradient UHPLC-SRM-MS analysis for the other three microorganisms. For the 25 minutes LC run, peptides were loaded to the column equilibrated with 5% B and hold for 0.6 minutes, followed by a linear gradient elution to 35% B over 20.4 minutes. The column was washed at 80% B for two minutes, and then equilibrated to 5% B for 1.5 minutes before loading next sample. For the 30 minutes LC run, peptides were loaded to the column equilibrated with 4% B and hold for 1 minutes, followed by a linear gradient elution to 40% B over 20 minutes. The column was washed at 90% B for 2 minutes, and then equilibrated to 4% B for 4 minutes before loading next sample.

Escherichia coli strain BW25113 (JPUB_001327), Saccharomyces cerevisiae S288C (JPUB_013514), Rhodosporidium toruloides NP11 (JPUB_012600), and Pseudomonas putida KT2440 (JPUB_012605) were cultured in house under the following conditions. For shake flask culturing at 200 RPM, E. coli and P. putida strains were grown for 24 hours in Luria broth (LB) medium at 37 °C and 30 °C respectively. S. cerevisiae and Rhodosporidium toruloides strains were grown overnight in YPD medium at 30ºC. Cells were distributed to 96 well plate format and harvested by centrifugation. Cell pellets were frozen at -80 °C until further processing either with the automated sample preparation workflow or manually.