Optimising timsTOF Pro 2 Mass Spectrometer Sample Preparation

Abstract

Varjosalo Research laboratory recently acquired a timsTOF Pro 2 mass spectrometer from Bruker. This instrument provides a much higher sensitivity than other mass spectrometers in the laboratory, thus optimising sample preparation became necessary. The goal of this project was to decrease the amount of starting material, i.e. the number of cells needed for a sample meant for analysis with the timsTOF Pro 2, and to determine suitable amounts of purification materials for sample preparation. Based on the original affinity purification sample preparation protocol (published in Nature Protocols in 2020) two modified protocols were designed. These protocols were protocol 3+2, which was used for samples made from three or two cell plates, and protocol 1 for samples made from one cell plate.

First, a trial run of three biological replicates purified from 1–5 cell plates was produced. The number of required interactors needed for a successful sample was decided from the five-plate samples diluted 1:20 according to the original protocol. For the other cell plate numbers, the trial samples were used to determine suitable resuspension volumes and dilutions to meet the criteria set by the five-plate samples. Results from four cell plates did not differ significantly from the three-plate sample results, thus no more four-plate samples were made. A new batch of samples was produced with six biological replicates for 1–3 and five cell plates to test for reproducibility and to allow for better statistical comparison of the identification results.

Protocol 3+2 made with three cell plates with a 1:4 dilution yielded the best overall results. According to statistical analysis the three-plate sample peptide spectrum match (PSM) values did not have statistically significant difference to the five-plate sample (original protocol) PSM values on a 95 % confidence level. Thus, it was concluded that the new protocol 3+2 produced with three cell plates per biological replicate, instead of the original five plates, is the best option for future affinity purification sample analysis with the timsTOF Pro 2. Protocol 3+2 also decreases the amount of purification materials needed for sample preparation, decreasing the overall cost of producing affinity purification samples.