High-Throughput Proteomics Enabled by a Fully Automated Dual-Trap and Dual-Column LC-MS.

This Technical Note describes a dual-column liquid chromatography system coupled to mass spectrometry (LC-MS) for high-throughput bottom-up proteomic analysis. This system made full use of two 2-position 10-port valves and a binary pump with an integrated loading pump of a commercial LC instrument to provide successive operation of two parallel subsystems. Each subsystem consisted of a set of trap columns and an analytical column. A T-junction union was used to split the mobile phase from the loading pump into two parts. This allowed one set of columns to be washed and equilibrated, followed by the injection of the next sample, while the previous sample was eluting and being analyzed on the other set of columns, thereby greatly increasing the analysis throughput. This approach showed high reproducibility for the analysis of HeLa tryptic digests with average relative standard deviation (RSD) values of 1.75%, 6.90%, and 5.19% for the identification number of proteins, peptides, and peptide-spectrum matches (PSMs), respectively, across 10 consecutive runs. The capacity for peptide and protein identification, as well as proteome depth, of the dual-column LC system was comparable to a conventional single-column system. Due to its simple equipment requirements and set up process, this method should be highly accessible for other laboratories.

Engineering an Oxygen-Binding Protein for Photocatalytic CO2 Reductions in Water.

While native CO2 -reducing enzymes display remarkable catalytic efficiency and product selectivity, few artificial biocatalysts have been engineered to allow understanding how the native enzymes work. To address this issue, we report cobalt porphyrin substituted myoglobin (CoMb) as a homogeneous catalyst for photo-driven CO2 to CO conversion in water. The activity and product selectivity were optimized by varying pH and concentrations of the enzyme and the photosensitizer. Up to 2000 TON(CO) was attained at low enzyme concentrations with low product selectivity (15 %), while a product selectivity of 74 % was reached by increasing the enzyme loading but with a compromised TON(CO). The efficiency of CO generation and overall TON(CO) were further improved by introducing positively charged residues (Lys or Arg) near the active stie of CoMb, which demonstrates the value of tuning the enzyme secondary coordination sphere to enhance the CO2 -reducing performance of a protein-based photocatalytic system.