Evaluation and optimization of quantitative mass spec sample prep workflows

Introduction: With the increase in proteomic sample preparation, instrumentation, and development of improved software tools, the number of biological studies utilizing Mass Spec data has increased in the last 5-10 years. Workflows including whole proteome screening after small molecule treatment (i.e. PROTACS, inhibitors, etc.), Immunoprecipitation, and PTM enrichment are commonly utilized. As these workflows are implemented, optimization of specific steps is needed. In this work we will focus on several workflows: PROTAC and kinase inhibitor treatment of cancer cells, enrichment of HiBiT tagged proteins, PTM enrichment (ubiquitin and phosphorylation), enrichment of exosomes, and automation. Improvements to specific steps in these workflows was examined with a focus on improvements to lysis bufferm proteolysis, desalting (i.e. detergent and surfactant removal), and steps to reduce time and increase overall workflow efficiency.

Methods: Human cancer cells were lysed with various lysis buffers desalted (SPE, precipitation, non-specific capture on magnetic beads, etc) and subjected to proteolysis. PTM"s or epitope tags were analyzed using magnetic beads specific for the appropriate target. Samples were analysed by nanoflow LC-MS/MS analysis using a capillary C18 column (0.075 x 250 mm; Acclaim PEPmap) coupled to an Exploris 240 Mass Spectrometer (Thermo) using both DDA and DIA methods. Data were processed using Byonic (Protein Metrics), PD 3.1 (Thermo) or Maxquant (Max Planck Institute).

Results: In our efforts to optimize our ability to quantify thousands of proteins in a single Mass Spec experiment, several factors were considered. These include selection of optimal lysis buffer, optimization of proteolysis, and desalting methods. In our evaluation , we have found that surfactant based lysis buffers produce more proteins and peptides relative to denaturant and solvent based lysis. Implementation of a sequential trypsin, Lys-c, and Arg-c Ultra protocol, in small ratios (1:10 versus 1:50) results in improved proteolytic efficiency at both lysine and arginine. With regard to desalting, we find that the use of magnetic beads, regardless of bead surface, performs as well or better than precipitation or filter-based methods, but with increased efficiency and without any technical challenges. This observation is particularly applicable for small samples (i.e. less than 5000 cells) where precipitation and filtration based methods are particularly challenging. These optimization steps were applied to the multiple workflows described above, resulting in improved analytical results (i.e. more quantifiable proteins) produced in shorter time periods. In many cases, because of the properties of magnetic beads, we can automate many of the workflows under study.