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The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of  in gel ,  on filter , and  in solution  digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The  in gel  methods interrogated were cost effective, but were limited in throughput and digest efficiency.  Filter-aided  sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two  in solution  digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent  RapiGest  led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion,  RapidACN , scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides.

The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics