Step-Wise Assessment and Optimization of Sample Handling Recovery Yield for Nanoproteomic Analysis of 1000 Mammalian Cells

Protein and peptide adhesion is a major factor contributing to sample loss during proteomic sample preparation workflows. Sample loss often has detrimental effects on the quality of proteomic analysis by compromising protein identification and data reproducibility. When starting with a low sample amount, only the most abundant proteins can be identified, which often offers little insights for biological research. Although the general idea about severe sample loss from low amount of starting material is widely presumed in the proteomics field, quantitative assessment on the impact of sample loss has been poorly investigated. In the present study, we have quantitatively assessed sample loss during each step of a conventional in-solution sample preparation workflow using bicinchoninic acid (BCA) and targeted LC/MS/MS protein and peptide assays. According to our assessment, for starting materials of ∼1000 mammalian cells, surface adhesion, along with desalting and speed-vacuum drying steps, all contribute heavily to sample loss, in particular for low-abundance proteins. With this knowledge, we have adapted slippery liquid infused porous surface (SLIPS) treatment, commercial LoBind tubes, and in-line desalting during sample processing. With these improvements, we were able to use a conventional in-solution sample handling method to identify on average 829 proteins with 1000 U2OS osteosarcoma cells (∼100 ng) with 75-min LC/MS/MS runs, an 11-fold increase in protein identification. Our optimized in-solution workflow is straightforward and also much less equipment- and technique-demanding than other advanced sample preparation protocols in the field.