Comparison of In-Gel and SP3 Based Sample Preparation Protocols for LC-MS/MS Based Proteomics.

Sensitivity, robustness, and reproducibility of sample preparation are main determinants of data quality in bottom-up mass spectrometry-based proteomics. To this end, in-gel protein clean-up and digestion has been used for decades and is characterized by its robustness and compatibility with harsh lysis conditions. Single-pot solid-phase-enhanced sample preparation (SP3) has gained substantial popularity recently and has been widely adapted as a standard workflow often replacing in-gel digestion-based workflows. Noteworthy, until today no direct comparison between the two workflows has been conducted. Here, we performed a systematic comparison of in-gel and SP3 based sample preparation workflows assessing sensitivity, robustness, reproducibility, and fractionation possibilities on human cellular lysates and blood plasma. Both methods performed similarly regarding number of identified proteins, however, showed specific biases. SP3 outperformed the in-gel workflow regarding higher sensitivity when handling limited sample amounts, especially below 5 µg of input material. In contrast, in-gel sample preparation was superior in the identification of low molecular weight proteins. In conclusion, while SP3 is indeed the state-of-the-art proteomics sample preparation method, in-gel digestion can deliver competitive and complementary results and still has advantages in some applications, such as measurement of small proteins or when there is a need for protein-level separation, e. g. in plasma samples.